Joint oligonucleotide, adaptor oligonucleotide and sequencing library construction method

Abstract

The invention provides a linker oligonucleotide, an adaptor oligonucleotide and a sequencing library construction method, and belongs to the technical field of biology. By optimizing the adapter sequence and / or adapter sequence embedded by the transposase complex in the transposase library building process, the loss caused by traditional transposase library building can be reduced.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and specifically relates to an adapter oligonucleotide, a linker oligonucleotide, and a method for constructing a sequencing library. Background Art

[0002] Next-generation Sequencing (NGS) has been widely used in fields such as genomic sequencing, disease diagnosis, and transcriptome research. When constructing a next-generation sequencing library, DNA needs to be fragmented into appropriate lengths, and then adapter sequences that can be recognized by the sequencing matrix and sequencing primers are added to both ends of the DNA fragments. Compared with the traditional library construction process, the Tn5 transposase method combines the fragmentation, end repair, and adapter ligation of DNA molecules into one reaction, with a short experimental process and convenient operation, greatly saving time. Since the probability of incorporating two adapter sequences, adapter1 and adapter2, into the ends of DNA fragments by using Tn5 is equal, there will be four situations at both ends of the DNA fragments in the product, namely adapter1+adapter1, adapter1+adapter2, adapter2+adapter1, and adapter2+adapter2. Only when the two ends of the DNA molecule contain different adapter sequences can it be correctly amplified and sequenced. Therefore, the theoretical yield will be lost by 50%.

[0003] Chinese Patent CN109154013B discloses a complex formed by a transposase and a Y linker for fragmenting and tagging DNA. After the Y linker sequences are inserted at both ends of the DNA, the gaps are filled by polymerase under non-strand displacement conditions, and ligation is used to complete the stitching. Alternatively, the downstream double-stranded region is partially displaced by a strand displacement polymerase, and the resulting structure can be eliminated by flap endonuclease, and ligation is used to complete the gap stitching, thereby generating a library with different adapter sequences at both ends to make up for the 50% loss.

[0004] Chinese Patent CN115552035A discloses a strategy of combining a transposase and a linker into a complex and then replacing the adapter. First, DNA-damaged nucleotides are used to reduce the polymerase activity to terminate strand displacement, and then adapter replacement is used to complete the terminal sequence replacement and addition, generating a target nucleic acid library labeled with both a forward adapter and a reverse adapter to avoid a 50% loss.

[0005] However, the inventors of the present application found that during the actual application of the complex synthesized by the Y adaptor, nucleotide strand displacement could not be completely avoided under non-strand displacement conditions, and it was not only a small number of nucleotides that underwent strand displacement under strand displacement conditions. The actual strand displacement process had a certain degree of uncontrollability, and the P7 sequence could be completely displaced while the polymerase extended to generate the complementary sequence of the P5 sequence. Therefore, the P5 sequence was inserted at both ends of the DNA, resulting in incorrect amplification. This method only partially compensated for the 50% loss of DNA, and there was still room for improvement in the library yield.

[0006] In addition, the use of the adaptor replacement strategy theoretically avoided the library loss caused by the uncontrollability of the above-mentioned strand displacement process. However, the overall library construction process involved multiple operations of opening the lid to add reagents and mixing, with complex operations, long processes and time-consuming, and relatively large losses in library output. Summary of the Invention

[0007] The purpose of the present application is to provide an adaptor oligonucleotide, a linker oligonucleotide, and a sequencing library construction. The method of the present application can solve the problem of 50% loss of sample DNA fragments during the construction of the library by traditional Tn5 transposase by optimizing the adaptor sequence embedded in the transposase and designing corresponding linkers, and improve the library construction output.

[0008] The first aspect of the present application provides an adaptor oligonucleotide, which comprises a first strand and a second strand. Wherein, the first strand comprises an optional upstream sequencing solid-phase binding sequence, an optional upstream sequencing primer sequence, and a transposase recognition core sequence from the 5' end to the 3' end, and the second strand comprises the reverse complementary sequence of an optional downstream sequencing solid-phase binding sequence, the reverse complementary sequence of an optional downstream sequencing primer sequence, and the reverse complementary sequence of the transposase recognition core sequence from the 3' end to the 5' end.

[0009] Wherein, one or more T bases in the transposase recognition core sequence of the first strand are replaced with damaged bases.

[0010] In some embodiments, the 1st or the 1st and 2nd or the 1st, 2nd and 3rd T bases from the 3' end in the transposase recognition core sequence of the first strand are replaced with damaged bases.

[0011] In some embodiments, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and abasic site, preferably U base.

[0012] In some embodiments, the methylated base is any one of, for example, N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymine; and the oxidized base is any one of, for example, 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA, and Fapy-dG.

[0013] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase, and further optionally, it is the ME sequence, and further optionally, it is as shown in SEQ ID NO.23.

[0014] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform, and further optionally, it is P5 or P7 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.24 or 25.

[0015] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform, and further optionally, it is P7 or P5 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.25 or 24.

[0016] In some embodiments, the upstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform, and further optionally, it is read1 or read2 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.26 or 27.

[0017] In some embodiments, the downstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform, and further optionally, it is read2 or read1 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.27 or 26.

[0018] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform, and further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform, and further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0019] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform, and further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform, and further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0020] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform. Further optionally, it is a first-strand sequencing primer or a second-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0021] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform. Further optionally, it is a first-strand sequencing primer or a second-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0022] In some embodiments, the adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence does not reverse complement when present, and the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence does not reverse complement when present.

[0023] In some embodiments, the first strand further comprises an upstream tag sequence, which is located at the 5' end or 3' end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3' end or 5' end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0024] In some embodiments, the adapter oligonucleotide may add an adapter spacer sequence at the 5' end of the ME sequence, especially when U is close to the 5' end; correspondingly, the linker oligonucleotide needs to add a corresponding sequence, and at this time, pay attention to the competition between the adapter spacer sequence and the linker spacer sequence.

[0025] In some embodiments, the first strand further comprises an adapter spacer sequence, which is located at the 5' end of the transposase recognition core sequence. Further optionally, the second strand further comprises the reverse complementary sequence of the adapter spacer sequence, which is located at the 3' end of the reverse complementary sequence of the transposase recognition core sequence. The adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence have a length of at least 1, 2, 3, 4, 5, 6 or more nucleotides.

[0026] A second aspect of the present application provides an adapter oligonucleotide comprising a first strand and a second strand, wherein the first strand comprises a transposase recognition core sequence and the second strand comprises the reverse complementary sequence of the transposase recognition core sequence.

[0027] Wherein, one or more T bases in the transposase recognition core sequence of the first strand are replaced with damaged bases.

[0028] In some embodiments, the 1st or the 1st and 2nd or the 1st, 2nd and 3rd T bases from the 3'-end in the transposase recognition core sequence of the first strand are replaced with damaged bases.

[0029] In some embodiments, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base and abasic site, preferably U base.

[0030] In some embodiments, the methylated base is any one of, for example, N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine and O4-methylthymine, the alkylated base is, for example, O6-alkylguanine or O4-alkylthymine, and the oxidized base is any one of, for example, 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA and Fapy-dG.

[0031] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase, and further optionally, it is the ME sequence, and further optionally, it is as shown in SEQ ID NO.23.

[0032] A third aspect of the present application provides an adapter oligonucleotide comprising a first strand and a second strand, wherein the first strand comprises an upstream sequencing primer sequence and a transposase recognition core sequence from the 5'-end to the 3'-end, and the second strand comprises the reverse complementary sequence of an optionally downstream sequencing primer sequence and the reverse complementary sequence of the transposase recognition core sequence from the 3'-end to the 5'-end.

[0033] Wherein, one or more T bases in the transposase recognition core sequence of the first strand are replaced with damaged bases.

[0034] In some embodiments, the 1st or the 1st and 2nd or the 1st, 2nd and 3rd T bases from the 3'-end in the transposase recognition core sequence of the first strand are replaced with damaged bases.

[0035] In some embodiments, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base and abasic site, preferably U base.

[0036] In some embodiments, the methylated base is any one of, for example, N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymine; and the oxidized base is any one of, for example, 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA, and Fapy-dG.

[0037] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase, and further optionally, it is the ME sequence, and further optionally, it is as shown in SEQ ID NO.23.

[0038] In some embodiments, the upstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform, and further optionally, it is read1 or read2 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.26 or 27.

[0039] In some embodiments, the downstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform, and further optionally, it is read2 or read1 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.27 or 26.

[0040] In some embodiments, the upstream sequencing primer sequence is the sequencing primer sequence of the MGI platform, and further optionally, it is the single-strand sequencing primer or double-strand sequencing primer of the MGI platform, and further optionally, it is Read1 or Read2 of the MGI platform.

[0041] In some embodiments, the downstream sequencing primer sequence is the sequencing primer sequence of the MGI platform, and further optionally, it is the single-strand sequencing primer or double-strand sequencing primer of the MGI platform, and further optionally, it is Read1 or Read2 of the MGI platform.

[0042] In some embodiments, the adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the upstream sequencing primer sequence and the reverse complementary sequence of the downstream sequencing primer sequence do not reverse complement when present.

[0043] In some embodiments, the first strand further comprises an upstream tag sequence located at the 5' end or 3' end of the upstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence located at the 3' end or 5' end of the reverse complementary sequence of the downstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0044] The fourth aspect of the present application provides an adapter oligonucleotide comprising a first strand and a second strand, wherein the first strand comprises an upstream sequencing primer sequence and a transposase recognition core sequence from the 5' end to the 3' end, and the second strand comprises the reverse complementary sequence of the downstream sequencing primer sequence and the reverse complementary sequence of the transposase recognition core sequence from the 3' end to the 5' end.

[0045] Wherein one or more T bases in the transposase recognition core sequence of the first strand are replaced with damaged bases.

[0046] Optionally, the first or the first and second or the first, second and third T bases from the 3' end in the transposase recognition core sequence of the first strand are replaced with damaged bases.

[0047] Optionally, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base and abasic site, preferably U base.

[0048] In some embodiments, the methylated base is any one of, for example, N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine and O4-methylthymine, the alkylated base is, for example, O6-alkylguanine or O4-alkylthymine, and the oxidized base is any one of, for example, 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA and Fapy-dG.

[0049] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase, further optionally, it is the ME sequence, and further optionally, it is as shown in SEQ ID NO.23.

[0050] In some embodiments, the upstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform, further optionally, it is read1 or read2 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.26 or 27.

[0051] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26.

[0052] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform. Further optionally, it is a single-strand sequencing primer or a double-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0053] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform. Further optionally, it is a single-strand sequencing primer or a double-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0054] In some embodiments, the adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence is not reverse complementary.

[0055] In some embodiments, the first strand further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3'-end or 5'-end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0056] The fifth aspect of the present application provides an adapter oligonucleotide, which comprises a first strand and a second strand. Wherein, the first strand comprises an upstream sequencing primer sequence and a transposase recognition core sequence from the 5'-end to the 3'-end, and the second strand comprises the reverse complementary sequence of the transposase recognition core sequence.

[0057] Wherein, one or more T bases in the transposase recognition core sequence of the first strand are replaced with damaged bases.

[0058] In some embodiments, the 1st or the 1st and 2nd or the 1st, 2nd and 3rd T bases from the 3'-end in the transposase recognition core sequence of the first strand are replaced with damaged bases.

[0059] In some embodiments, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and abasic site, preferably the U base.

[0060] In some embodiments, the methylated base is any one of, for example, N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymine, the alkylated base is, for example, O6-alkylguanine or O4-alkylthymine, and the oxidized base is any one of, for example, 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA, and Fapy-dG.

[0061] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase, and further optionally, it is the ME sequence, and further optionally, it is as shown in SEQ ID NO.23.

[0062] In some embodiments, the upstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform, and further optionally, it is read1 or read2 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.26 or 27.

[0063] In some embodiments, the upstream sequencing primer sequence is the sequencing primer sequence of the MGI platform, and further optionally, it is the single-strand sequencing primer or double-strand sequencing primer of the MGI platform, and further optionally, it is Read1 or Read2 of the MGI platform.

[0064] In some embodiments, the first strand further comprises an upstream tag sequence, which is located at the 5' end or 3' end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0065] The sixth aspect of the present application provides an adapter oligonucleotide, which comprises a first strand and a second strand. Wherein, the first strand comprises an upstream sequencing solid-phase binding sequence, an upstream sequencing primer sequence, and a transposase recognition core sequence from the 5' end to the 3' end, and the second strand comprises, from the 3' end to the 5' end, the reverse complementary sequence of an optionally downstream sequencing solid-phase binding sequence, the reverse complementary sequence of an optionally downstream sequencing primer sequence, and the reverse complementary sequence of the transposase recognition core sequence.

[0066] Wherein, one or more T bases in the transposase recognition core sequence of the first strand are replaced with damaged bases.

[0067] In some embodiments, the 1st or the 1st and 2nd or the 1st, 2nd and 3rd T bases from the 3'-end in the transposase recognition core sequence of the first strand are replaced with damaged bases.

[0068] In some embodiments, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base and abasic site, preferably U base.

[0069] In some embodiments, the methylated base is any one of, for example, N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine and O4-methylthymine, the alkylated base is, for example, O6-alkylguanine or O4-alkylthymine, and the oxidized base is any one of, for example, 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA and Fapy-dG.

[0070] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase, and further optionally, it is the ME sequence, and further optionally, it is as shown in SEQ ID NO.23.

[0071] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform, and further optionally, it is P5 or P7 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.24 or 25.

[0072] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform, and further optionally, it is P7 or P5 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.25 or 24.

[0073] In some embodiments, the upstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform, and further optionally, it is read1 or read2 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.26 or 27.

[0074] In some embodiments, the downstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform, and further optionally, it is read2 or read1 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.27 or 26.

[0075] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0076] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0077] In some embodiments, the upstream sequencing primer sequence is the sequencing primer sequence of the MGI platform. Further optionally, it is the first-strand sequencing primer or the second-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0078] In some embodiments, the downstream sequencing primer sequence is the sequencing primer sequence of the MGI platform. Further optionally, it is the first-strand sequencing primer or the second-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0079] In some embodiments, the adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence does not reverse complement when present, and the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence does not reverse complement when present.

[0080] In some embodiments, the first strand further comprises an upstream tag sequence, which is located at the 5' end or the 3' end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3' end or the 5' end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0081] The seventh aspect of the present application provides an adapter oligonucleotide, which comprises a first strand and a second strand. Wherein, the first strand comprises an upstream sequencing solid-phase binding sequence, an upstream sequencing primer sequence, and a transposase recognition core sequence from the 5' end to the 3' end, and the second strand comprises a reverse complementary sequence of a downstream sequencing solid-phase binding sequence, a reverse complementary sequence of a downstream sequencing primer sequence, and a reverse complementary sequence of the transposase recognition core sequence from the 3' end to the 5' end.

[0082] Wherein, one or more T bases in the transposase recognition core sequence of the first strand are replaced with damaged bases.

[0083] In some embodiments, the 1st or the 1st and 2nd or the 1st, 2nd and 3rd T bases from the 3' end in the transposase recognition core sequence of the first strand are replaced with damaged bases.

[0084] In some embodiments, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and abasic site, preferably U base.

[0085] In some embodiments, the methylated base is any one of, for example, N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymine, the alkylated base is, for example, O6-alkylguanine or O4-alkylthymine, and the oxidized base is any one of, for example, 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA, and Fapy-dG.

[0086] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase. Further optionally, it is an ME sequence. Further optionally, it is as shown in SEQ ID NO.23.

[0087] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25.

[0088] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24.

[0089] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27.

[0090] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26.

[0091] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0092] In some embodiments, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0093] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform. Further optionally, it is the single-strand sequencing primer or the double-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0094] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform. Further optionally, it is the single-strand sequencing primer or the double-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0095] In some embodiments, the adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence is not reverse complementary, and the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence is not reverse complementary.

[0096] In some embodiments, the first strand further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the reverse complementary sequence of the downstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0097] The eighth aspect of the present application provides an adapter oligonucleotide comprising a first strand and a second strand, wherein the first strand comprises an upstream sequencing solid-phase binding sequence, an upstream sequencing primer sequence and a transposase recognition core sequence from the 5' end to the 3' end, and the second strand comprises the reverse complementary sequence of the transposase recognition core sequence.

[0098] Wherein one or more T bases in the transposase recognition core sequence of the first strand are replaced with damaged bases.

[0099] In some embodiments, the 1st or the 1st and 2nd or the 1st, 2nd and 3rd T bases from the 3' end in the transposase recognition core sequence of the first strand are replaced with damaged bases.

[0100] In some embodiments, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base and abasic site, preferably U base.

[0101] In some embodiments, the methylated base is any one of, for example, N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine and O4-methylthymine, the alkylated base is, for example, O6-alkylguanine or O4-alkylthymine, and the oxidized base is any one of, for example, 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA and Fapy-dG.

[0102] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase, and further optionally, it is the ME sequence, and further optionally, it is as shown in SEQ ID NO.23.

[0103] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform, and further optionally, it is P5 or P7 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.24 or 25.

[0104] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27.

[0105] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0106] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform. Further optionally, it is the first-strand sequencing primer or the second-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0107] In some embodiments, the first strand further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0108] The ninth aspect of the present application provides an adapter oligonucleotide, which comprises an optional first strand and a second strand. Wherein, the first strand comprises, from the 5'-end to the 3'-end, an optional upstream sequencing solid-phase binding sequence, an upstream sequencing primer sequence and an adapter spacer sequence. The second strand comprises, from the 3'-end to the 5'-end, the reverse complementary sequence of an optional downstream sequencing solid-phase binding sequence, the reverse complementary sequence of a downstream sequencing primer sequence, the reverse complementary sequence of an optional said adapter spacer sequence, the 3'-end portion of the reverse complementary sequence of a transposase recognition core sequence, and an A base.

[0109] Wherein, the 3'-end portion of the reverse complementary sequence of the transposase recognition core sequence is reverse complementary to the 5'-end portion of the transposase recognition core sequence of the first strand of the linker oligonucleotide according to the first aspect. Wherein, the 5'-end portion of the transposase recognition core sequence refers to the portion of the transposase recognition core sequence at the 5'-end of the damaged base.

[0110] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25.

[0111] In some embodiments, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24.

[0112] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27.

[0113] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26.

[0114] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0115] In some embodiments, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0116] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform. Further optionally, it is the single-strand sequencing primer or the double-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0117] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform. Further optionally, it is the single-strand sequencing primer or the double-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0118] In some embodiments, the adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence does not reverse complement when present, and the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence does not reverse complement when present.

[0119] In some embodiments, the first strand further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the reverse complementary sequence of the downstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, wherein the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0120] The tenth aspect of the present application provides an adapter oligonucleotide comprising a first strand and a second strand, wherein the first strand, from the 5' end to the 3' end, comprises an optional upstream sequencing solid-phase binding sequence, an upstream sequencing primer sequence and an adapter spacer sequence, and the second strand, from the 3' end to the 5' end, comprises the reverse complementary sequence of an optional downstream sequencing solid-phase binding sequence, the reverse complementary sequence of a downstream sequencing primer sequence, the reverse complementary sequence of the adapter spacer sequence, the 3' end portion of the reverse complementary sequence of the transposase recognition core sequence and an A base.

[0121] Wherein, the 3' end portion of the reverse complementary sequence of the transposase recognition core sequence is reverse complementary to the 5' end portion of the transposase recognition core sequence of the first strand of the linker oligonucleotide according to the second aspect, and the 5' end portion of the transposase recognition core sequence refers to the portion of the transposase recognition core sequence at the 5' end of the damaged base.

[0122] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P5 or P7 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.24 or 25.

[0123] In some embodiments, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P7 or P5 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.25 or 24.

[0124] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, further optionally, it is read1 or read2 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.26 or 27.

[0125] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence for the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26.

[0126] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence for the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0127] In some embodiments, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence for the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0128] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence for the MGI platform. Further optionally, it is the single-strand sequencing primer or the double-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0129] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence for the MGI platform. Further optionally, it is the single-strand sequencing primer or the double-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0130] In some embodiments, the adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence does not reverse complement when present, and the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence does not reverse complement when present.

[0131] In some embodiments, the first strand further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3'-end or 5'-end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, wherein the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0132] The eleventh invention of the present application provides an adapter oligonucleotide, which comprises a first strand and a second strand. The first strand comprises an upstream sequencing primer sequence and an adapter spacer sequence from the 5'-end to the 3'-end, and the second strand comprises the reverse complementary sequence of a downstream sequencing primer sequence, the reverse complementary sequence of the adapter spacer sequence, the 3'-end portion of the reverse complementary sequence of the transposase recognition core sequence and an A base from the 3'-end to the 5'-end.

[0133] Wherein, the 3'-end portion of the reverse complementary sequence of the transposase recognition core sequence is reverse complementary to the 5'-end portion of the transposase recognition core sequence of the first strand of the linker oligonucleotide according to the second aspect, and the 5'-end portion of the transposase recognition core sequence refers to the portion of the transposase recognition core sequence at the 5'-end of the damaged base.

[0134] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27.

[0135] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26.

[0136] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform. Further optionally, it is a single-strand sequencing primer or a double-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0137] In some embodiments, the downstream sequencing primer sequence is the sequencing primer sequence of the MGI platform. Further optionally, it is the first-strand sequencing primer or the second-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0138] In some embodiments, the adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary.

[0139] Optionally, the first strand further comprises an upstream tag sequence, which is located at the 5' end or the 3' end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3' end or the 5' end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. Among them, the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0140] The twelfth invention of the present application provides an adapter oligonucleotide, which comprises a first strand and a second strand. Among them, the first strand comprises an upstream sequencing solid-phase binding sequence, an upstream sequencing primer sequence and an adapter spacer sequence from the 5' end to the 3' end. The second strand comprises the reverse complementary sequence of a downstream sequencing solid-phase binding sequence, the reverse complementary sequence of a downstream sequencing primer sequence, the reverse complementary sequence of the adapter spacer sequence, the 3' end portion of the reverse complementary sequence of the transposase recognition core sequence and an A base from the 3' end to the 5' end.

[0141] Among them, the 3' end portion of the reverse complementary sequence of the transposase recognition core sequence is reverse complementary to the 5' end portion of the transposase recognition core sequence of the first strand of the linker oligonucleotide according to the second aspect. Among them, the 5' end portion of the transposase recognition core sequence refers to the portion of the transposase recognition core sequence at the 5' end of the damaged base.

[0142] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25.

[0143] In some embodiments, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24.

[0144] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27.

[0145] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26.

[0146] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0147] In some embodiments, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0148] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform. Further optionally, it is the single-strand sequencing primer or the double-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0149] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform. Further optionally, it is the single-strand sequencing primer or the double-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0150] In some embodiments, the adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence is not reverse complementary, and the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence is not reverse complementary.

[0151] In some embodiments, the first strand further comprises an upstream tag sequence located at the 5'-end or 3'-end of the upstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence located at the 3'-end or 5'-end of the reverse complementary sequence of the downstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, wherein the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0152] The thirteenth aspect of the present application provides an adapter oligonucleotide comprising a second strand, wherein the second strand from the 3'-end to the 5'-end comprises the reverse complementary sequence of the downstream sequencing primer sequence, the 3'-end portion of the reverse complementary sequence of the transposase recognition core sequence, and an A base.

[0153] Wherein, the 3'-end portion of the reverse complementary sequence of the transposase recognition core sequence is reverse complementary to the 5'-end portion of the transposase recognition core sequence of the first strand of the linker oligonucleotide according to the fifth aspect, wherein the 5'-end portion of the transposase recognition core sequence refers to the portion of the transposase recognition core sequence at the 5'-end of the damaged base.

[0154] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and further optionally, it is read2 or read1 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.27 or 26.

[0155] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and further optionally, it is a single-strand sequencing primer or a double-strand sequencing primer of the MGI platform, and further optionally, it is Read1 or Read2 of the MGI platform.

[0156] In some embodiments, the reverse complementary sequence of the downstream sequencing primer sequence is not reverse complementary to the upstream sequencing primer sequence of the linker oligonucleotide according to the fifth aspect.

[0157] In some embodiments, the second strand further comprises the reverse complementary sequence of a downstream tag sequence located at the 3'-end or 5'-end of the reverse complementary sequence of the downstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0158] The fourteenth aspect of the present application provides an adapter oligonucleotide comprising a second strand, wherein the second strand from the 3'-end to the 5'-end comprises the reverse complementary sequence of the downstream sequencing solid-phase binding sequence, the reverse complementary sequence of the downstream sequencing primer sequence, the 3'-end portion of the reverse complementary sequence of the transposase recognition core sequence, and an A base.

[0159] Wherein, the 3'-end portion of the reverse complementary sequence of the transposase recognition core sequence is reverse complementary to the 5'-end portion of the transposase recognition core sequence of the first strand of the adapter oligonucleotide according to the eighth aspect, and the 5'-end portion of the transposase recognition core sequence refers to the portion of the transposase recognition core sequence at the 5'-end of the damaged base.

[0160] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P7 or P5 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.25 or 24.

[0161] In some embodiments, the downstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform, further optionally, it is read2 or read1 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.27 or 26.

[0162] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform, further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform, and further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0163] In some embodiments, the downstream sequencing primer sequence is the sequencing primer sequence of the MGI platform, further optionally, it is the single-strand sequencing primer or the double-strand sequencing primer of the MGI platform, and further optionally, it is Read1 or Read2 of the MGI platform.

[0164] In some embodiments, the reverse complementary sequence of the downstream sequencing primer sequence is not reverse complementary to the upstream sequencing primer sequence of the adapter oligonucleotide according to the eighth aspect, and the reverse complementary sequence of the downstream sequencing solid-phase binding sequence is not reverse complementary to the upstream sequencing solid-phase binding sequence of the adapter oligonucleotide according to the eighth aspect.

[0165] In some embodiments, the second strand further comprises a reverse complementary sequence of a downstream tag sequence, which is located at the 3' end or 5' end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0166] The fifteenth aspect of the present application provides a method for constructing a sequencing library, which includes: (1) using a transposase complex embedded with the adapter oligonucleotide according to the first aspect to fragment the target sequence; (2) treating with a DNA polymerase intolerant to damaged bases, FEN1 enzyme and ligase, optionally adding the adapter oligonucleotide according to the ninth aspect before ligase treatment and performing denaturation and annealing; and (3) optionally performing PCR using an extension primer pair,

[0167] wherein, the upstream primer of the extension primer pair comprises an upstream sequencing solid-phase binding sequence and the upstream sequencing primer sequence from the 5' end to the 3' end, and the downstream primer of the extension primer pair comprises a downstream sequencing solid-phase binding sequence and the downstream sequencing primer sequence from the 5' end to the 3' end.

[0168] In some embodiments, the DNA polymerase intolerant to damaged bases is one or more of DNA polymerases intolerant to U bases, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase and Tgo DNA polymerase.

[0169] In some embodiments, the DNA polymerase intolerant to damaged bases is Pfu DNA polymerase or Deep vent DNA polymerase intolerant to hypoxanthine.

[0170] In some embodiments, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase and Taq DNA ligase.

[0171] In some embodiments, the FEN1 enzyme includes, but is not limited to, the wild type, mutants, homologous proteins and isozymes of the FEN1 enzyme, such as Rad27 / FEN1 and Rad2 / FEN1.

[0172] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P5 or P7 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.24 or 25.

[0173] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24.

[0174] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0175] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0176] In some embodiments, the upstream sequencing solid-phase binding sequence and the reverse complementary sequence of the downstream sequencing solid-phase binding sequence do not reverse complement when present.

[0177] In some embodiments, the upstream primer further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the downstream primer further comprises a downstream tag sequence, which is located at the 5'-end or 3'-end of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0178] The sixteenth aspect of the present application provides a method for constructing a sequencing library, which includes: (1) using a transposase complex embedded with the adapter oligonucleotide according to the second aspect to fragment the target sequence; (2) using a DNA polymerase intolerant to damaged bases, FEN1 enzyme and ligase for treatment, wherein the adapter oligonucleotide according to the eleventh aspect is added and denatured and annealed before the ligase treatment; and (3) performing PCR using an extension primer pair.

[0179] Wherein, the upstream primer of the extension primer pair comprises an upstream sequencing solid-phase binding sequence and the upstream sequencing primer sequence from the 5'-end to the 3'-end, and the downstream primer of the extension primer pair comprises a downstream sequencing solid-phase binding sequence and the downstream sequencing primer sequence from the 5'-end to the 3'-end.

[0180] In some embodiments, the DNA polymerase intolerant to damaged bases is one or more of the DNA polymerases intolerant to U bases, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, and Tgo DNA polymerase.

[0181] In some embodiments, the DNA polymerase intolerant to damaged bases is Pfu DNA polymerase or Deep vent DNA polymerase intolerant to hypoxanthine.

[0182] In some embodiments, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, and Taq DNA ligase.

[0183] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25.

[0184] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24.

[0185] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0186] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0187] In some embodiments, when present, the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence is not reverse complementary.

[0188] In some embodiments, the upstream primer further comprises an upstream tag sequence located at the 5'-end or 3'-end of the upstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the downstream primer further comprises a downstream tag sequence located at the 5'-end or 3'-end of the downstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0189] The seventeenth aspect of the present application provides a method for constructing a sequencing library, which includes: (1) using a transposase complex encapsulated with the adaptor oligonucleotide according to the second aspect to fragment the target sequence; and (2) treating with a DNA polymerase intolerant to damaged bases, FEN1 enzyme and ligase, wherein the adaptor oligonucleotide according to the twelfth aspect is added and denatured and annealed before the ligase treatment.

[0190] In some embodiments, the DNA polymerase intolerant to damaged bases is one or more of the DNA polymerases intolerant to U base, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase and Tgo DNA polymerase.

[0191] In some embodiments, the DNA polymerase intolerant to damaged bases is PfuDNA polymerase or Deep vent DNA polymerase intolerant to hypoxanthine.

[0192] In some embodiments, the FEN1 enzyme includes, but is not limited to, the wild type, mutants, homologous proteins and isozymes of the FEN1 enzyme, such as Rad27 / FEN1 and Rad2 / FEN1.

[0193] In some embodiments, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase and Taq DNA ligase.

[0194] The eighteenth aspect of the present application provides a method for constructing a sequencing library, which includes: (1) using a transposase complex encapsulated with the adaptor oligonucleotide according to the fourth aspect to fragment the target sequence; (2) treating with a DNA polymerase intolerant to damaged bases, FEN1 enzyme and ligase; and (3) performing PCR using extension primer pairs.

[0195] Among them, the upstream primer of the extension primer pair contains an upstream sequencing solid-phase binding sequence and the upstream sequencing primer sequence from the 5'-end to the 3'-end, and the downstream primer of the extension primer pair contains a downstream sequencing solid-phase binding sequence and the downstream sequencing primer sequence from the 5'-end to the 3'-end.

[0196] In some embodiments, the DNA polymerase intolerant to damaged bases is one or more of the DNA polymerases intolerant to U base, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase and Tgo DNA polymerase.

[0197] In some embodiments, the DNA polymerase intolerant to damaged bases is Pfu DNA polymerase or Deep vent DNA polymerase intolerant to hypoxanthine.

[0198] In some embodiments, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase and Taq DNA ligase.

[0199] In some embodiments, the polymerase intolerant to damaged bases is used to fill in the 9-base gap existing between the adapter oligonucleotide and the target sequence, followed by strand displacement. When the strand displacement reaction proceeds to the damaged base site, it stops, generating a forked double-stranded DNA structure. The FEN1 enzyme is used to excise the 5' Flap structure in the forked double-stranded DNA structure to generate a gap, and the ligase is used to complete the gap ligation, so that different adapter sequences are carried at both ends of the target sequence, as Figure 9 shown.

[0200] In some embodiments, the FEN1 enzyme includes but is not limited to the wild type, mutant, homologous protein and isoenzyme of the FEN1 enzyme, such as Rad27 / FEN1 and Rad2 / FEN1.

[0201] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is shown as SEQ ID NO.24 or 25.

[0202] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is shown as SEQ ID NO.25 or 24.

[0203] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0204] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0205] In some embodiments, the upstream sequencing solid-phase binding sequence and the reverse complementary sequence of the downstream sequencing solid-phase binding sequence, when present, are not reverse complementary.

[0206] In some embodiments, the upstream primer further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. And / or, the downstream primer further comprises a downstream tag sequence, which is located at the 5'-end or 3'-end of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0207] The nineteenth aspect of the present application provides a method for constructing a sequencing library, which includes: (1) using a transposase complex encapsulated with the adapter oligonucleotide according to the fifth aspect to fragment the target sequence; (2) treating with a DNA polymerase intolerant to damaged bases, FEN1 enzyme and ligase, wherein the adapter oligonucleotide according to the thirteenth aspect is added and denatured and annealed before the ligase treatment; and (3) performing PCR using an extension primer pair.

[0208] Wherein, the upstream primer of the extension primer pair comprises an upstream sequencing solid-phase binding sequence and the upstream sequencing primer sequence from the 5'-end to the 3'-end, and the downstream primer of the extension primer pair comprises a downstream sequencing solid-phase binding sequence and the downstream sequencing primer sequence from the 5'-end to the 3'-end.

[0209] In some embodiments, the DNA polymerase intolerant to damaged bases is one or more of the DNA polymerases intolerant to U bases, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase and Tgo DNA polymerase.

[0210] In some embodiments, the DNA polymerase intolerant to damaged bases is Pfu DNA polymerase or Deep vent DNA polymerase intolerant to hypoxanthine.

[0211] Optionally, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, and Taq DNA ligase.

[0212] In some embodiments, the FEN1 enzyme includes, but is not limited to, the wild type, mutants, homologous proteins, and isozymes of the FEN1 enzyme, such as Rad27 / FEN1 and Rad2 / FEN1.

[0213] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25.

[0214] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24.

[0215] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0216] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0217] In some embodiments, the upstream sequencing solid-phase binding sequence and the reverse complementary sequence of the downstream sequencing solid-phase binding sequence, when present, are not reverse complementary.

[0218] In some embodiments, the upstream primer further comprises an upstream tag sequence located at the 5'-end or 3'-end of the upstream sequencing primer sequence and having a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the downstream primer further comprises a downstream tag sequence located at the 5'-end or 3'-end of the downstream sequencing primer sequence and having a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0219] The twentieth aspect of the present application provides a method for constructing a sequencing library, which includes: (1) using a transposase complex encapsulated with the adapter oligonucleotide according to the seventh aspect to fragment the target sequence; and (2) using a DNA polymerase intolerant to damaged bases, FEN1 enzyme and ligase for treatment.

[0220] In some embodiments, the DNA polymerase intolerant to damaged bases is one or more of DNA polymerases intolerant to U base, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase and Tgo DNA polymerase.

[0221] In some embodiments, the DNA polymerase intolerant to damaged bases is Pfu DNA polymerase or Deep vent DNA polymerase intolerant to hypoxanthine.

[0222] In some embodiments, the DNA polymerase intolerant to damaged bases is Pfu DNA polymerase or Deep vent DNA polymerase intolerant to hypoxanthine.

[0223] In some embodiments, the polymerase intolerant to damaged bases is used to fill in the 9-base gap existing between the adapter oligonucleotide and the target sequence, followed by strand displacement, which is stopped when the strand displacement reaction proceeds to the damaged base site, generating a branched double-stranded DNA structure. The FEN1 enzyme is used to excise the 5' Flap structure in the branched double-stranded DNA structure to generate a gap, and the ligase is used to complete the gap ligation, so that different adapter sequences are carried at both ends of the target sequence.

[0224] In some embodiments, the FEN1 enzyme includes but is not limited to the wild type, mutant, homologous protein and isoenzyme of the FEN1 enzyme, such as Rad27 / FEN1 and Rad2 / FEN1.

[0225] In some embodiments, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, and Taq DNA ligase.

[0226] The twenty - first aspect of the present application provides a method for constructing a sequencing library, which includes: (1) using a transposase complex embedded with the adapter oligonucleotide according to the eighth aspect to fragment the target sequence; and (2) treating with a DNA polymerase intolerant to loss, FEN1 enzyme, and ligase, wherein the adapter oligonucleotide according to the fourteenth aspect is added and denatured and annealed before the ligase treatment.

[0227] In some embodiments, the DNA polymerase intolerant to damaged bases is one or more of the DNA polymerases intolerant to U base, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase, and Tgo DNA polymerase.

[0228] In some embodiments, the DNA polymerase intolerant to damaged bases is Pfu DNA polymerase or Deep vent DNA polymerase intolerant to hypoxanthine.

[0229] In some embodiments, the DNA polymerase intolerant to damaged bases is Pfu DNA polymerase or Deep vent DNA polymerase intolerant to hypoxanthine.

[0230] In some embodiments, the FEN1 enzyme includes, but is not limited to, the wild - type, mutants, homologous proteins, and isozymes of the FEN1 enzyme, such as Rad27 / FEN1 and Rad2 / FEN1.

[0231] In some embodiments, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, and Taq DNA ligase.

[0232] The twenty - second aspect of the present application provides a kit, which contains

[0233] (1) The adapter oligonucleotide according to the second aspect, the adapter oligonucleotide according to the eleventh aspect, and the extension primer pair;

[0234] (2) The adapter oligonucleotide according to the second aspect and the adapter oligonucleotide according to the twelfth aspect;

[0235] (3) The adapter oligonucleotide according to the fourth aspect and the extension primer pair;

[0236] (4) The adapter oligonucleotide according to the fifth aspect, the linker oligonucleotide according to the thirteenth aspect, and the extension primer pair;

[0237] (5) The adapter oligonucleotide according to the seventh aspect; or

[0238] (6) The adapter oligonucleotide according to the eighth aspect and the linker oligonucleotide according to the fourteenth aspect,

[0239] wherein, the upstream primer of the extension primer pair includes an upstream sequencing solid-phase binding sequence and the upstream sequencing primer sequence from the 5'-end to the 3'-end, and the downstream primer of the extension primer pair includes a downstream sequencing solid-phase binding sequence and the downstream sequencing primer sequence from the 5'-end to the 3'-end.

[0240] In some embodiments, the upstream primer further includes an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or, the downstream primer further includes a downstream tag sequence, which is located at the 5'-end or 3'-end of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0241] In some embodiments, the kit further includes one or more of the following: (1) transposase; (2) DNA polymerase intolerant to damaged bases; (3) FEN1 enzyme; and (4) ligase.

[0242] In some embodiments, the DNA polymerase intolerant to damaged bases is one or more of DNA polymerases intolerant to U base, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, and Tgo DNA polymerase.

[0243] In some embodiments, the DNA polymerase intolerant to damaged bases is PfuDNA polymerase or Deep vent DNA polymerase intolerant to hypoxanthine.

[0244] In some embodiments, the FEN1 enzyme includes, but is not limited to, the wild type, mutants, homologous proteins, and isozymes of the FEN1 enzyme, such as Rad27 / FEN1 and Rad2 / FEN1.

[0245] In some embodiments, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, and Taq DNA ligase.

[0246] The twenty-third aspect of the present application provides an adapter oligonucleotide, which comprises a first strand and a second strand. Wherein, the first strand comprises, from the 5'-end to the 3'-end, an optional upstream sequencing solid-phase binding sequence, an optional upstream sequencing primer sequence, an adapter spacer sequence, one or more damaged bases, and a transposase recognition core sequence; the second strand comprises, from the 3'-end to the 5'-end, the reverse complementary sequence of an optional downstream sequencing solid-phase binding sequence, the reverse complementary sequence of an optional downstream sequencing primer sequence, the reverse complementary sequence of an optional said adapter spacer sequence, an optional one or more A bases, and the reverse complementary sequence of the transposase recognition core sequence.

[0247] Wherein, the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0248] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase, further optionally, it is an ME sequence, and further optionally, it is as shown in SEQ ID NO.23.

[0249] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P5 or P7 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.24 or 25.

[0250] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P7 or P5 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.25 or 24.

[0251] In some embodiments, the upstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform, further optionally, it is read1 or read2 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.26 or 27.

[0252] In some embodiments, the downstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform, further optionally, it is read2 or read1 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.27 or 26.

[0253] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0254] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0255] In some embodiments, the upstream sequencing primer sequence is the sequencing primer sequence of the MGI platform. Further optionally, it is the first-strand sequencing primer or the second-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0256] In some embodiments, the downstream sequencing primer sequence is the sequencing primer sequence of the MGI platform. Further optionally, it is the first-strand sequencing primer or the second-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0257] In some embodiments, the adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence does not reverse complement when present, and the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence does not reverse complement when present.

[0258] In some embodiments, the first strand further comprises an upstream tag sequence, which is located at the 5' end or the 3' end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3' end or the 5' end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0259] In some embodiments, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base and abasic site, preferably U base.

[0260] In some embodiments, the methylated base is any one of, for example, N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymine; and the oxidized base is any one of, for example, 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA, and Fapy-dG.

[0261] In some embodiments, the number of damaged bases is at most 20, preferably at most 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and the number of A bases is the same as the number of damaged bases.

[0262] In some embodiments, the adapter oligonucleotide can add an adapter spacer sequence to the 5'-end of the ME sequence.

[0263] In some embodiments, the first strand further comprises an adapter spacer sequence located at the 5'-end of the transposase recognition core sequence. Optionally, the second strand further comprises the reverse complementary sequence of the adapter spacer sequence, and the reverse complementary sequence of the adapter spacer sequence is located at the 3'-end of the reverse complementary sequence of the transposase recognition core sequence. The adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence are at least 1, 2, 3, 4, 5, 6, or more nucleotides in length.

[0264] The twenty-fourth aspect of the present application provides an adapter oligonucleotide comprising a first strand and a second strand. The first strand, from the 5'-end to the 3'-end, comprises an adapter spacer sequence, a damaged base, and a transposase recognition core sequence. The second strand, from the 3'-end to the 5'-end, comprises, optionally, the reverse complementary sequence of the adapter spacer sequence, optionally an A base, and the reverse complementary sequence of the transposase recognition core sequence.

[0265] Wherein, the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence are at least 1, 2, 3, 4, 5, 6, 7, 8, or more nucleotides in length.

[0266] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase, optionally further an ME sequence, and optionally further as shown in SEQ ID NO.23.

[0267] In some embodiments, the damaged base is any one of a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and abasic site, preferably a U base.

[0268] In some embodiments, the methylated base is any one of, for example, N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymine; and the oxidized base is any one of, for example, 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA, and Fapy-dG.

[0269] The twenty-fifth aspect of the present application provides an adapter oligonucleotide, which comprises a first strand and a second strand. Wherein, the first strand comprises an adapter spacer sequence, a damaged base, and a transposase recognition core sequence from the 5'-end to the 3'-end; the second strand comprises the reverse complementary sequence of the adapter spacer sequence, an A base, and the reverse complementary sequence of the transposase recognition core sequence from the 3'-end to the 5'-end.

[0270] Wherein, the lengths of the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence are at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0271] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase, and further optionally, it is an ME sequence, and further optionally, it is as shown in SEQ ID NO.23.

[0272] In some embodiments, the damaged base is any one of a U base, hypoxanthine, xanthine, a methylated base, an oxidized base, an alkylated base, and an abasic site, preferably a U base.

[0273] In some embodiments, the methylated base is any one of, for example, N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymine; and the oxidized base is any one of, for example, 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA, and Fapy-dG.

[0274] The twenty-sixth aspect of the present application provides an adapter oligonucleotide, which comprises a first strand and a second strand. Wherein, the first strand comprises an adapter spacer sequence, a damaged base, and a transposase recognition core sequence from the 5'-end to the 3'-end; the second strand comprises the reverse complementary sequence of the transposase recognition core sequence.

[0275] Wherein, the length of the adapter spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0276] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase. Further optionally, it is the ME sequence. Further optionally, it is as shown in SEQ ID NO.23.

[0277] In some embodiments, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and abasic site, preferably the U base.

[0278] In some embodiments, the methylated base is any one of, for example, N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymine; and the oxidized base is any one of, for example, 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA, and Fapy-dG.

[0279] A second aspect of the present application provides an adapter oligonucleotide, which comprises a first strand and a second strand. Wherein, the first strand comprises an upstream sequencing primer sequence, an adapter spacer sequence, a damaged base, and a transposase recognition core sequence from the 5' end to the 3' end; the second strand comprises, from the 3' end to the 5' end, the reverse complementary sequence of an optionally downstream sequencing primer sequence, the reverse complementary sequence of the optionally adapter spacer sequence, an optionally A base, and the reverse complementary sequence of the transposase recognition core sequence.

[0280] Wherein, the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0281] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase. Further optionally, it is the ME sequence. Further optionally, it is as shown in SEQ ID NO.23.

[0282] In some embodiments, the upstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27.

[0283] In some embodiments, the downstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26.

[0284] In some embodiments, the upstream sequencing primer sequence is the sequencing primer sequence of the MGI platform. Further optionally, it is the first-strand sequencing primer or the second-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0285] In some embodiments, the downstream sequencing primer sequence is the sequencing primer sequence of the MGI platform. Further optionally, it is the first-strand sequencing primer or the second-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0286] In some embodiments, the adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence does not reverse complement when present.

[0287] In some embodiments, the first strand further comprises an upstream tag sequence, which is located at the 5' end or the 3' end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3' end or the 5' end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0288] In some embodiments, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base and abasic site, preferably U base.

[0289] In some embodiments, the methylated base is any one of, for example, N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine and O4-methylthymine, the alkylated base is, for example, O6-alkylguanine or O4-alkylthymine, and the oxidized base is any one of, for example, 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA and Fapy-dG.

[0290] The twenty-eighth aspect of the present application provides an adapter oligonucleotide, which comprises a first strand and a second strand. Wherein, the first strand comprises an upstream sequencing primer sequence, an adapter spacer sequence, a damaged base and a transposase recognition core sequence from the 5' end to the 3' end, and the second strand comprises the reverse complementary sequence of the downstream sequencing primer sequence, the reverse complementary sequence of the adapter spacer sequence, an A base and the reverse complementary sequence of the transposase recognition core sequence from the 3' end to the 5' end.

[0291] Wherein, the length of the linker spacer sequence and the reverse complementary sequence of the linker spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0292] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase, further optionally, it is the ME sequence, and further optionally, it is as shown in SEQ ID NO.23.

[0293] In some embodiments, the upstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform, further optionally, it is read1 or read2 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.26 or 27.

[0294] In some embodiments, the downstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform, further optionally, it is read2 or read1 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.27 or 26.

[0295] In some embodiments, the upstream sequencing primer sequence is the sequencing primer sequence of the MGI platform, further optionally, it is the single-strand sequencing primer or double-strand sequencing primer of the MGI platform, and further optionally, it is Read1 or Read2 of the MGI platform.

[0296] In some embodiments, the downstream sequencing primer sequence is the sequencing primer sequence of the MGI platform, further optionally, it is the single-strand sequencing primer or double-strand sequencing primer of the MGI platform, and further optionally, it is Read1 or Read2 of the MGI platform.

[0297] In some embodiments, the linker oligonucleotide is a Y-shaped linker oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence is not reverse complementary.

[0298] In some embodiments, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base and abasic site, and preferably the U base.

[0299] In some embodiments, the methylated base is any one of, for example, N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine and O4-methylthymine, the alkylated base is, for example, O6-alkylguanine or O4-alkylthymine, and the oxidized base is, for example, any one of 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA and Fapy-dG.

[0300] In some embodiments, the first strand further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises a reverse complementary sequence of a downstream tag sequence, which is located at the 3'-end or 5'-end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0301] The twenty-ninth aspect of the present application provides an adapter oligonucleotide, which comprises a first strand and a second strand. The first strand comprises an upstream sequencing primer sequence, an adapter spacer sequence, a damaged base and a transposase recognition core sequence from the 5'-end to the 3'-end, and the second strand comprises a reverse complementary sequence of the transposase recognition core sequence.

[0302] Wherein, the length of the adapter spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0303] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase, further optionally, it is an ME sequence, and further optionally, it is as shown in SEQ ID NO.23.

[0304] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, further optionally, it is read1 or read2 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.26 or 27.

[0305] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, further optionally, it is a single-strand sequencing primer or a double-strand sequencing primer of the MGI platform, and further optionally, it is Read1 or Read2 of the MGI platform.

[0306] In some embodiments, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base and abasic site, and preferably U base.

[0307] In some embodiments, the methylated base is any one of, for example, N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymine; and the oxidized base is any one of, for example, 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA, and Fapy-dG.

[0308] In some embodiments, the first strand further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0309] The thirtieth aspect of the present application provides an adapter oligonucleotide, which comprises a first strand and a second strand. Wherein, the first strand comprises, from the 5'-end to the 3'-end, an upstream sequencing solid-phase binding sequence, an upstream sequencing primer sequence, an adapter spacer sequence, a damaged base, and a transposase recognition core sequence; the second strand comprises, from the 3'-end to the 5'-end, the reverse complementary sequence of an optional downstream sequencing solid-phase binding sequence, the reverse complementary sequence of an optional downstream sequencing primer sequence, the reverse complementary sequence of an optional said adapter spacer sequence, an optional A base, and the reverse complementary sequence of the transposase recognition core sequence.

[0310] Wherein, the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0311] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase, further optionally, it is an ME sequence, and further optionally, it is as shown in SEQ ID NO.23.

[0312] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P5 or P7 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.24 or 25.

[0313] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P7 or P5 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.25 or 24.

[0314] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27.

[0315] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26.

[0316] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0317] In some embodiments, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0318] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform. Further optionally, it is the single-strand sequencing primer or the double-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0319] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform. Further optionally, it is the single-strand sequencing primer or the double-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0320] In some embodiments, the adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence does not reverse complement when present, and the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence does not reverse complement when present.

[0321] In some embodiments, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base and abasic site, preferably U base.

[0322] In some embodiments, the methylated base is any one of, for example, N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymine; and the oxidized base is any one of, for example, 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA, and Fapy-dG.

[0323] In some embodiments, the first strand further comprises an upstream tag sequence located at the 5'-end or 3'-end of the upstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence located at the 3'-end or 5'-end of the reverse complementary sequence of the downstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more nucleotides.

[0324] The thirty-first aspect of the present application provides an adapter oligonucleotide comprising a first strand and a second strand. The first strand, from the 5'-end to the 3'-end, comprises an upstream sequencing solid-phase binding sequence, an upstream sequencing primer sequence, an adapter spacer sequence, a damaged base, and a transposase recognition core sequence. The second strand, from the 3'-end to the 5'-end, comprises the reverse complementary sequence of a downstream sequencing solid-phase binding sequence, the reverse complementary sequence of a downstream sequencing primer sequence, the reverse complementary sequence of the adapter spacer sequence, an A base, and the reverse complementary sequence of the transposase recognition core sequence.

[0325] Wherein, the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8, or more nucleotides.

[0326] Optionally, the transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase. Further optionally, it is the ME sequence. Further optionally, it is as shown in SEQ ID NO.23.

[0327] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25.

[0328] In some embodiments, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24.

[0329] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27.

[0330] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26.

[0331] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0332] In some embodiments, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0333] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform. Further optionally, it is the single-strand sequencing primer or the double-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0334] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform. Further optionally, it is the single-strand sequencing primer or the double-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0335] In some embodiments, the adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence is not reverse complementary, and the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence is not reverse complementary.

[0336] In some embodiments, the damaged base is any one of a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and abasic site, preferably a U base.

[0337] In some embodiments, the methylated base is any one of, for example, N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymine; and the oxidized base is any one of, for example, 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA, and Fapy-dG.

[0338] In some embodiments, the first strand further comprises an upstream tag sequence located at the 5'-end or 3'-end of the upstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more nucleotides; and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence located at the 3'-end or 5'-end of the reverse complementary sequence of the downstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more nucleotides.

[0339] The thirty-second aspect of the present application provides an adapter oligonucleotide comprising a first strand and a second strand. The first strand, from the 5'-end to the 3'-end, comprises an upstream sequencing solid-phase binding sequence, an upstream sequencing primer sequence, an adapter spacer sequence, a damaged base, and a transposase recognition core sequence; the second strand comprises the reverse complementary sequence of the transposase recognition core sequence.

[0340] Wherein, the length of the adapter spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8, or more nucleotides.

[0341] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase, further optionally, it is an ME sequence, and further optionally, it is as shown in SEQ ID NO.23.

[0342] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P5 or P7 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.24 or 25.

[0343] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27.

[0344] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence or the vesicular sequence of the vesicle adapter of the MGI platform.

[0345] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform. Further optionally, it is the first-strand sequencing primer or the second-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0346] In some embodiments, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and abasic site, preferably U base.

[0347] In some embodiments, the methylated base is any one of, for example, N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymine. The alkylated base is, for example, O6-alkylguanine or O4-alkylthymine. The oxidized base is any one of, for example, 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA, and Fapy-dG.

[0348] In some embodiments, the first strand further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0349] The thirty-third aspect of the present application provides an adapter oligonucleotide, which comprises an optional first strand and a second strand. Wherein, the first strand comprises, from the 5'-end to the 3'-end, an optional upstream sequencing solid-phase binding sequence, an upstream sequencing primer sequence, and an adapter spacer sequence. The second strand comprises, from the 3'-end to the 5'-end, the reverse complementary sequence of an optional downstream sequencing solid-phase binding sequence, the reverse complementary sequence of a downstream sequencing primer sequence, the reverse complementary sequence of an optional said adapter spacer sequence, the reverse complementary sequence of a linker spacer sequence, and an A base.

[0350] Among them, the reverse complementary sequence of the adapter spacer sequence is reverse complementary to the adapter spacer sequence of the first strand of the adapter oligonucleotide according to the twenty-third aspect.

[0351] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25.

[0352] In some embodiments, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24.

[0353] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27.

[0354] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26.

[0355] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0356] In some embodiments, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0357] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform. Further optionally, it is the single-strand sequencing primer or the double-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0358] In some embodiments, the downstream sequencing primer sequence is the sequencing primer sequence of the MGI platform. Further optionally, it is the first-strand sequencing primer or the second-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0359] In some embodiments, the adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence does not reverse complement when present, and the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence does not reverse complement when present.

[0360] In some embodiments, the first strand further comprises an upstream tag sequence, which is located at the 5' end or the 3' end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3' end or the 5' end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0361] In some embodiments, the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0362] The thirty-fourth aspect of the present application provides an adapter oligonucleotide, which comprises a first strand and a second strand. Wherein, the first strand comprises, from the 5' end to the 3' end, optionally an upstream sequencing solid-phase binding sequence, an upstream sequencing primer sequence, and an adapter spacer sequence; the second strand comprises, from the 3' end to the 5' end, the reverse complementary sequence of an optionally downstream sequencing solid-phase binding sequence, the reverse complementary sequence of a downstream sequencing primer sequence, the reverse complementary sequence of the adapter spacer sequence, the reverse complementary sequence of a linker spacer sequence, and an A base, wherein the reverse complementary sequence of the linker spacer sequence is reverse complementary to the linker spacer sequence of the first strand of the linker oligonucleotide according to the twenty-fourth aspect.

[0363] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25.

[0364] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24.

[0365] In some embodiments, the upstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27.

[0366] In some embodiments, the downstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26.

[0367] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0368] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0369] In some embodiments, the upstream sequencing primer sequence is the sequencing primer sequence of the MGI platform. Further optionally, it is the single-strand sequencing primer or the double-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0370] In some embodiments, the downstream sequencing primer sequence is the sequencing primer sequence of the MGI platform. Further optionally, it is the single-strand sequencing primer or the double-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0371] In some embodiments, the adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence does not reverse complement when present, and the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence does not reverse complement when present.

[0372] In some embodiments, the first strand further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the reverse complementary sequence of the downstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0373] In some embodiments, the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0374] The thirty-fifth aspect of the present application provides an adapter oligonucleotide comprising a first strand and a second strand, wherein the first strand comprises an upstream sequencing primer sequence and an adapter spacer sequence from the 5' end to the 3' end, and the second strand comprises the reverse complementary sequence of the downstream sequencing primer sequence, the reverse complementary sequence of the adapter spacer sequence, the reverse complementary sequence of the linker spacer sequence and an A base from the 3' end to the 5' end.

[0375] Wherein, the reverse complementary sequence of the linker spacer sequence is reverse complementary to the linker spacer sequence of the first strand of the linker oligonucleotide according to the twenty-sixth aspect.

[0376] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, further optionally, it is read1 or read2 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.26 or 27.

[0377] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, further optionally, it is read2 or read1 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.27 or 26.

[0378] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, further optionally, it is a single-strand sequencing primer or a double-strand sequencing primer of the MGI platform, and further optionally, it is Read1 or Read2 of the MGI platform.

[0379] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence for the MGI platform. Further optionally, it is a first-strand sequencing primer or a second-strand sequencing primer for the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0380] In some embodiments, the adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the upstream sequencing primer sequence are not reverse complementary.

[0381] In some embodiments, the first strand further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3'-end or 5'-end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0382] In some embodiments, the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0383] The thirty-sixth aspect of the present application provides an adapter oligonucleotide, which comprises a first strand and a second strand. Wherein, the first strand from the 5'-end to the 3'-end comprises an upstream sequencing solid-phase binding sequence, an upstream sequencing primer sequence and an adapter spacer sequence, and the second strand from the 3'-end to the 5'-end comprises the reverse complementary sequence of a downstream sequencing solid-phase binding sequence, the reverse complementary sequence of a downstream sequencing primer sequence, the reverse complementary sequence of the adapter spacer sequence, the reverse complementary sequence of a linker spacer sequence and an A base,

[0384] Wherein, the reverse complementary sequence of the linker spacer sequence is reverse complementary to the linker spacer sequence of the first strand of the linker oligonucleotide according to the twenty-sixth aspect.

[0385] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence for the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25.

[0386] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24.

[0387] In some embodiments, the upstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27.

[0388] In some embodiments, the downstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26.

[0389] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0390] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0391] In some embodiments, the upstream sequencing primer sequence is the sequencing primer sequence of the MGI platform. Further optionally, it is the single-strand sequencing primer or the double-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0392] In some embodiments, the downstream sequencing primer sequence is the sequencing primer sequence of the MGI platform. Further optionally, it is the single-strand sequencing primer or the double-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0393] In some embodiments, the adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence is not reverse complementary, and the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence is not reverse complementary.

[0394] In some embodiments, the first strand further comprises an upstream tag sequence, which is located at the 5' or 3' end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3' or 5' end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0395] In some embodiments, the adaptor spacer sequence and the reverse complementary sequence of the adaptor spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0396] The thirty-seventh aspect of the present application provides an adaptor oligonucleotide, which comprises a second strand, wherein the second strand comprises, from the 3' end to the 5' end, the reverse complementary sequence of a downstream sequencing primer sequence, the reverse complementary sequence of a linker spacer sequence, and an A base.

[0397] Wherein, the reverse complementary sequence of the linker spacer sequence is reverse complementary to the linker spacer sequence of the first strand of the adaptor oligonucleotide according to the twenty-ninth aspect.

[0398] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and further optionally, it is read2 or read1 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.27 or 26.

[0399] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and further optionally, it is a single-strand sequencing primer or a double-strand sequencing primer of the MGI platform, and further optionally, it is Read1 or Read2 of the MGI platform.

[0400] In some embodiments, the reverse complementary sequence of the downstream sequencing primer sequence is not reverse complementary to the upstream sequencing primer sequence of the adaptor oligonucleotide according to the twenty-ninth aspect.

[0401] In some embodiments, the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3' or 5' end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0402] The thirty-eighth aspect of the present application provides an adapter oligonucleotide, which comprises a second strand, wherein the second strand comprises, from the 3'-end to the 5'-end, the reverse complementary sequence of the downstream sequencing solid-phase binding sequence, the reverse complementary sequence of the downstream sequencing primer sequence, the reverse complementary sequence of the linker spacer sequence, and an A base.

[0403] Wherein, the reverse complementary sequence of the linker spacer sequence is reverse complementary to the linker spacer sequence of the first strand of the linker oligonucleotide according to the thirty-second aspect.

[0404] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform, and further optionally, it is P7 or P5 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.25 or 24.

[0405] In some embodiments, the downstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform, and further optionally, it is read2 or read1 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.27 or 26.

[0406] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform, and further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform, and further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0407] In some embodiments, the downstream sequencing primer sequence is the sequencing primer sequence of the MGI platform, and further optionally, it is the single-strand sequencing primer or the double-strand sequencing primer of the MGI platform, and further optionally, it is Read1 or Read2 of the MGI platform.

[0408] In some embodiments, the reverse complementary sequence of the downstream sequencing primer sequence is not reverse complementary to the upstream sequencing primer sequence of the linker oligonucleotide according to the thirty-second aspect, and the reverse complementary sequence of the downstream sequencing solid-phase binding sequence is not reverse complementary to the upstream sequencing solid-phase binding sequence of the linker oligonucleotide according to the thirty-second aspect.

[0409] In some embodiments, the second strand further comprises the reverse complementary sequence of the downstream tag sequence, and the downstream tag sequence is located at the 3'-end or 5'-end of the reverse complementary sequence of the downstream sequencing primer sequence, and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0410] The thirty-ninth aspect of the present application provides a method for constructing a sequencing library, which includes: (1) using a transposase complex embedded with the adapter oligonucleotide according to the twenty-third aspect to fragment the target sequence; (2) treating with a DNA polymerase and a ligase that are intolerant to damaged bases, and optionally, adding the adapter oligonucleotide according to the thirty-third aspect before ligase treatment and performing denaturation and annealing; and (3) optionally, performing PCR using an extension primer pair,

[0411] wherein, the upstream primer of the extension primer pair includes an upstream sequencing solid-phase binding sequence and the upstream sequencing primer sequence from the 5'-end to the 3'-end, and the downstream primer of the extension primer pair includes a downstream sequencing solid-phase binding sequence and the downstream sequencing primer sequence from the 5'-end to the 3'-end.

[0412] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P5 or P7 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.24 or 25.

[0413] In some embodiments, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P7 or P5 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.25 or 24.

[0414] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the MGI platform, further optionally, it is an upstream splint adapter sequence or a downstream splint adapter sequence of the MGI platform, and further optionally, it is a linear sequence of the vesicle adapter or a vesicular sequence of the vesicle adapter of the MGI platform.

[0415] In some embodiments, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the MGI platform, further optionally, it is an upstream splint adapter sequence or a downstream splint adapter sequence of the MGI platform, and further optionally, it is a linear sequence of the vesicle adapter or a vesicular sequence of the vesicle adapter of the MGI platform.

[0416] In some embodiments, the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence, when present, is not reverse complementary.

[0417] In some embodiments, the DNA polymerase that is intolerant to damaged bases is one or more of the DNA polymerases that are intolerant to U bases, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase, and Tgo DNA polymerase.

[0418] In some embodiments, the DNA polymerase that is intolerant to damaged bases is Pfu DNA polymerase or Deep vent DNA polymerase that is intolerant to hypoxanthine.

[0419] In some embodiments, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, and Taq DNA ligase.

[0420] In some embodiments, the upstream primer further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the downstream primer further comprises a downstream tag sequence, which is located at the 5'-end or 3'-end of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0421] The fortieth aspect of the present application provides a method for constructing a sequencing library, which includes: (1) using a transposase complex encapsulated with the adapter oligonucleotide according to the twenty-fifth aspect to fragment the target sequence; (2) treating with a DNA polymerase that is intolerant to damaged bases, FEN1 enzyme, and a ligase, wherein the adapter oligonucleotide according to the thirty-fifth aspect is added and denatured and annealed before the ligase treatment; and (3) performing PCR using an extension primer pair.

[0422] Wherein, the upstream primer of the extension primer pair comprises an upstream sequencing solid-phase binding sequence and the upstream sequencing primer sequence from the 5'-end to the 3'-end, and the downstream primer of the extension primer pair comprises a downstream sequencing solid-phase binding sequence and the downstream sequencing primer sequence from the 5'-end to the 3'-end.

[0423] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P5 or P7 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.24 or 25.

[0424] In some embodiments, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P7 or P5 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.25 or 24.

[0425] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform, and further optionally, is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform, and further optionally, is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0426] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform, and further optionally, is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform, and further optionally, is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0427] In some embodiments, the upstream sequencing solid-phase binding sequence and the reverse complementary sequence of the downstream sequencing solid-phase binding sequence, when present, are not reverse complementary.

[0428] In some embodiments, the DNA polymerase intolerant to damaged bases is one or more of the DNA polymerases intolerant to U bases, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase, and Tgo DNA polymerase.

[0429] In some embodiments, the DNA polymerase intolerant to damaged bases is Pfu DNA polymerase or Deep vent DNA polymerase intolerant to hypoxanthine.

[0430] In some embodiments, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, and Taq DNA ligase.

[0431] In some embodiments, the upstream primer further comprises an upstream tag sequence, the upstream tag sequence is located at the 5' end or the 3' end of the upstream sequencing primer sequence, and the length is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or, the downstream primer further comprises a downstream tag sequence, the downstream tag sequence is located at the 5' end or the 3' end of the downstream sequencing primer sequence, and the length is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0432] The forty - first aspect of the present application provides a method for constructing a sequencing library, which includes: (1) using a transposase complex embedded with the adapter oligonucleotide according to the twenty - fifth aspect to fragment the target sequence; and (2) treating with a DNA polymerase intolerant to damaged bases, FEN1 enzyme, and ligase, wherein the adapter oligonucleotide according to the thirty - sixth aspect is added and denatured and annealed before the ligase treatment.

[0433] In some embodiments, the DNA polymerase intolerant to damaged bases is one or more of the DNA polymerases intolerant to U base, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase, and Tgo DNA polymerase.

[0434] In some embodiments, the DNA polymerase intolerant to damaged bases is Pfu DNA polymerase or Deep vent DNA polymerase intolerant to hypoxanthine.

[0435] In some embodiments, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, and Taq DNA ligase.

[0436] The forty - second aspect of the present application provides a method for constructing a sequencing library, which includes: (1) using a transposase complex embedded with the adapter oligonucleotide according to the twenty - sixth aspect to fragment the target sequence; (2) treating with a DNA polymerase intolerant to damaged bases and a ligase, wherein the adapter oligonucleotide according to the thirty - fifth aspect is added before the ligase treatment; and (3) performing PCR using an extension primer pair.

[0437] Wherein, the upstream primer of the extension primer pair includes an upstream sequencing solid - phase binding sequence and the upstream sequencing primer sequence from the 5' end to the 3' end, and the downstream primer of the extension primer pair includes a downstream sequencing solid - phase binding sequence and the downstream sequencing primer sequence from the 5' end to the 3' end.

[0438] In some embodiments, the upstream sequencing solid - phase binding sequence is the sequencing solid - phase binding sequence of the Illumina platform, further optionally, it is P5 or P7 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.24 or 25.

[0439] In some embodiments, the downstream sequencing solid - phase binding sequence is the sequencing solid - phase binding sequence of the Illumina platform, further optionally, it is P7 or P5 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.25 or 24.

[0440] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0441] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0442] In some embodiments, the upstream sequencing solid-phase binding sequence and the reverse complementary sequence of the downstream sequencing solid-phase binding sequence, when present, are not reverse complementary.

[0443] In some embodiments, the DNA polymerase intolerant to damaged bases is one or more of the DNA polymerases intolerant to U base, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase and Tgo DNA polymerase.

[0444] In some embodiments, the DNA polymerase intolerant to damaged bases is Pfu DNA polymerase or Deep vent DNA polymerase intolerant to hypoxanthine.

[0445] In some embodiments, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase and Taq DNA ligase.

[0446] In some embodiments, the upstream primer further comprises an upstream tag sequence, which is located at the 5' end or 3' end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the downstream primer further comprises a downstream tag sequence, which is located at the 5' end or 3' end of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0447] The forty-third aspect of the present application provides a method for constructing a sequencing library, which includes: (1) using a transposase complex embedded with the adapter oligonucleotide according to the twenty-sixth aspect to fragment the target sequence; and (2) using a DNA polymerase and a ligase that are intolerant to damaged bases for treatment, wherein the adapter oligonucleotide according to the thirty-sixth aspect is added before the ligase treatment.

[0448] In some embodiments, the DNA polymerase that is intolerant to damaged bases is one or more of the DNA polymerases that are intolerant to U bases, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase, and Tgo DNA polymerase.

[0449] In some embodiments, the DNA polymerase that is intolerant to damaged bases is Pfu DNA polymerase or Deep vent DNA polymerase that is intolerant to hypoxanthine.

[0450] In some embodiments, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, and Taq DNA ligase.

[0451] The forty-fourth aspect of the present application provides a method for constructing a sequencing library, which includes: (1) using a transposase complex embedded with the adapter oligonucleotide according to the twenty-eighth aspect to fragment the target sequence; (2) using a DNA polymerase that is intolerant to damage, FEN1 enzyme, and ligase for treatment; and (3) performing PCR using an extension primer pair.

[0452] Wherein, the upstream primer of the extension primer pair includes an upstream sequencing solid-phase binding sequence and the upstream sequencing primer sequence from the 5'-end to the 3'-end, and the downstream primer of the extension primer pair includes a downstream sequencing solid-phase binding sequence and the downstream sequencing primer sequence from the 5'-end to the 3'-end.

[0453] In some embodiments, the DNA polymerase that is intolerant to damaged bases is one or more of the DNA polymerases that are intolerant to U bases, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase, and Tgo DNA polymerase.

[0454] In some embodiments, the DNA polymerase that is intolerant to damaged bases is Pfu DNA polymerase or Deep vent DNA polymerase that is intolerant to hypoxanthine.

[0455] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P5 or P7 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.24 or 25.

[0456] In some embodiments, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P7 or P5 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.25 or 24.

[0457] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the MGI platform, further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform, and further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0458] In some embodiments, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the MGI platform, further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform, and further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0459] In some embodiments, the upstream sequencing solid-phase binding sequence and the reverse complementary sequence of the downstream sequencing solid-phase binding sequence, when present, are not reverse complementary.

[0460] In some embodiments, the upstream primer further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or, the downstream primer further comprises a downstream tag sequence, which is located at the 5'-end or 3'-end of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0461] The forty-fifth aspect of the present application provides a method for constructing a sequencing library, which includes: (1) using a transposase complex embedded with the adapter oligonucleotide according to the twenty-ninth aspect to fragment the target sequence; (2) using a DNA polymerase and a ligase that are intolerant to damaged bases for treatment, wherein the adapter oligonucleotide according to the thirty-seventh aspect is added before the ligase treatment; and (3) performing PCR using extension primers.

[0462] Among them, the upstream primer of the extension primer pair includes an upstream sequencing solid-phase binding sequence and the upstream sequencing primer sequence from the 5'-end to the 3'-end, and the downstream primer of the extension primer pair includes a downstream sequencing solid-phase binding sequence and the downstream sequencing primer sequence from the 5'-end to the 3'-end.

[0463] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P5 or P7 of the Illumina platform, and further optionally, it is shown as SEQ ID NO.24 or 25.

[0464] In some embodiments, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P7 or P5 of the Illumina platform, and further optionally, it is shown as SEQ ID NO.25 or 24.

[0465] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the MGI platform, further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform, and further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0466] In some embodiments, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the MGI platform, further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform, and further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0467] In some embodiments, the upstream sequencing solid-phase binding sequence and the reverse complementary sequence of the downstream sequencing solid-phase binding sequence are not reverse complementary when present.

[0468] In some embodiments, the DNA polymerase intolerant to damaged bases is one or more of the DNA polymerases intolerant to U bases, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase, and Tgo DNA polymerase.

[0469] In some embodiments, the DNA polymerase intolerant to damaged bases is Pfu DNA polymerase or Deep vent DNA polymerase intolerant to hypoxanthine.

[0470] In some embodiments, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, and Taq DNA ligase.

[0471] In some embodiments, the upstream primer further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the downstream primer further comprises a downstream tag sequence, which is located at the 5'-end or 3'-end of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0472] The forty-sixth aspect of the present application provides a method for constructing a sequencing library, which includes: (1) using a transposase complex encapsulated with the adapter oligonucleotide according to the thirty-second aspect to fragment the target sequence; and (2) treating with a DNA polymerase intolerant to damaged bases, FEN1 enzyme and ligase.

[0473] In some embodiments, the DNA polymerase intolerant to damaged bases is one or more of the DNA polymerases intolerant to U base, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase and Tgo DNA polymerase.

[0474] In some embodiments, the DNA polymerase intolerant to damaged bases is Pfu DNA polymerase or Deep vent DNA polymerase intolerant to hypoxanthine.

[0475] In some embodiments, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase and Taq DNA ligase.

[0476] The forty-seventh aspect of the present application provides a method for constructing a sequencing library, which includes: (1) using a transposase complex encapsulated with the adapter oligonucleotide according to the thirty-second aspect to fragment the target sequence; and (2) treating with a DNA polymerase intolerant to damaged bases and a ligase, wherein the adapter oligonucleotide according to the thirty-eighth aspect is added before the ligase treatment.

[0477] In some embodiments, the DNA polymerase intolerant to damaged bases is one or more of the DNA polymerases intolerant to U base, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase and Tgo DNA polymerase.

[0478] In some embodiments, the DNA polymerase intolerant to damaged bases is Pfu DNA polymerase or Deep vent DNA polymerase intolerant to hypoxanthine.

[0479] In some embodiments, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, and Taq DNA ligase.

[0480] The forty-eighth aspect of the present application provides a kit, which comprises

[0481] (1) The adapter oligonucleotide according to the twenty-fifth aspect, the adaptor oligonucleotide according to the thirty-fifth aspect, and the extension primer pair;

[0482] (2) The adapter oligonucleotide according to the twenty-fifth aspect and the adaptor oligonucleotide according to the thirty-sixth aspect;

[0483] (3) The adapter oligonucleotide according to the twenty-sixth aspect, the adaptor oligonucleotide according to the thirty-fifth aspect, and the extension primer pair;

[0484] (4) The adapter oligonucleotide according to the twenty-sixth aspect and the adaptor oligonucleotide according to the thirty-sixth aspect;

[0485] (5) The adapter oligonucleotide according to the twenty-eighth aspect and the extension primer pair;

[0486] (6) The adapter oligonucleotide according to the twenty-ninth aspect, the adaptor oligonucleotide according to the thirty-seventh aspect, and the extension primer pair;

[0487] (7) The adapter oligonucleotide according to the thirty-first aspect; or

[0488] (8) The adapter oligonucleotide according to the thirty-second aspect and the adaptor oligonucleotide according to the thirty-eighth aspect,

[0489] wherein the upstream primer of the extension primer pair comprises an upstream sequencing solid-phase binding sequence and the upstream sequencing primer sequence from the 5'-end to the 3'-end, and the downstream primer of the extension primer pair comprises a downstream sequencing solid-phase binding sequence and the downstream sequencing primer sequence from the 5'-end to the 3'-end.

[0490] In some embodiments, the upstream primer further comprises an upstream tag sequence located at the 5' end or 3' end of the upstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the downstream primer further comprises a downstream tag sequence located at the 5' end or 3' end of the downstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0491] In some embodiments, the kit further comprises one or more of the following: (1) transposase; (2) a DNA polymerase intolerant to damaged bases; and (3) ligase.

[0492] Optionally, the DNA polymerase intolerant to damaged bases is one or more of the DNA polymerases intolerant to U base, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, and Tgo DNA polymerase.

[0493] In some embodiments, the DNA polymerase intolerant to damaged bases is Pfu DNA polymerase or Deep vent DNA polymerase intolerant to hypoxanthine.

[0494] In some embodiments, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, and Taq DNA ligase.

[0495] The forty-ninth aspect of the present application provides an adapter oligonucleotide comprising a first strand and a second strand. The first strand, from the 5' end to the 3' end, comprises an optional upstream sequencing solid-phase binding sequence, an optional upstream sequencing primer sequence, an optional adapter spacer sequence, one or more restriction sequences I, and a transposase recognition core sequence. The second strand, from the 3' end to the 5' end, comprises the reverse complementary sequence of an optional downstream sequencing solid-phase binding sequence, the reverse complementary sequence of an optional downstream sequencing primer sequence, the reverse complementary sequence of an optional adapter spacer sequence, one or more restriction sequences II, and the reverse complementary sequence of the transposase recognition core sequence. The restriction sequence I and the restriction sequence II constitute the recognition / cutting site of a restriction endonuclease.

[0496] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase. Further optionally, it is the ME sequence. Further optionally, it is as shown in SEQ ID NO. 23.

[0497] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25.

[0498] In some embodiments, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24.

[0499] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27.

[0500] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26.

[0501] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0502] In some embodiments, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0503] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform. Further optionally, it is the single-strand sequencing primer or the double-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0504] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform. Further optionally, it is the single-strand sequencing primer or the double-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0505] In some embodiments, the adapter oligonucleotide is a Y-shaped adapter oligonucleotide, i.e., the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence, when present, is not reverse complementary, and the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence, when present, is not reverse complementary.

[0506] In some embodiments, the first strand further comprises an upstream tag sequence located at the 5' end or 3' end of the upstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence located at the 3' end or 5' end of the reverse complementary sequence of the downstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0507] In some embodiments, the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0508] In some embodiments, the restriction enzyme is a restriction enzyme known to those of ordinary skill in the art that can recognize a restriction sequence and cut to produce sticky ends, including but not limited to EcoRI, HindIII, BamHI, XhoI, EcoRV, SalI, XbaI, PstI, SmaI, NotI, KpnI, SacI, SphI, EcoRII, BglII, AvaI, NdeI, SstI, BstEII, HpaI, BspEI, BspHI, BspQI, BsrBI, NlaIII, NlaIV, MlyI, DdeI, DpnI, MmeI, FokI, etc.

[0509] In some embodiments, the sticky end is a 5' sticky end or a 3' sticky end.

[0510] In some embodiments, the sticky end has at most 2, 3, 4, 5, 6, 7, 8, 9 or 10 protruding unpaired bases.

[0511] In some embodiments, the adapter oligonucleotide can add an adapter spacer sequence at the 5' end of the restriction site to improve the cleavage efficiency.

[0512] The fiftieth aspect of the present application provides an adapter oligonucleotide, which comprises a first strand and a second strand. Wherein, the first strand comprises, from the 5'-end to the 3'-end, an optional upstream sequencing solid-phase binding sequence, an upstream sequencing primer sequence, an optional adapter spacer sequence, an optional linker spacer sequence, and a restriction sequence III; the second strand comprises, from the 3'-end to the 5'-end, the reverse complementary sequence of an optional downstream sequencing solid-phase binding sequence, the reverse complementary sequence of a downstream sequencing primer sequence, the reverse complementary sequence of an optional said adapter spacer sequence, the reverse complementary sequence of an optional said linker spacer sequence, and a restriction sequence IV.

[0513] Wherein, the linker spacer sequence is the same as the linker spacer sequence described in the forty-ninth aspect.

[0514] Wherein, the restriction sequence III and the restriction sequence IV constitute the sticky ends after cleavage by the restriction endonuclease described in the forty-ninth aspect.

[0515] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25.

[0516] In some embodiments, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24.

[0517] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27.

[0518] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26.

[0519] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0520] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0521] In some embodiments, the upstream sequencing primer sequence is the sequencing primer sequence of the MGI platform. Further optionally, it is the first-strand sequencing primer or the second-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0522] In some embodiments, the downstream sequencing primer sequence is the sequencing primer sequence of the MGI platform. Further optionally, it is the first-strand sequencing primer or the second-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0523] In some embodiments, the adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence does not reverse complement when present, and the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence does not reverse complement when present.

[0524] In some embodiments, the first strand further comprises an upstream tag sequence, which is located at the 5' end or the 3' end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3' end or the 5' end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0525] In some embodiments, the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0526] The fifty-first aspect of the present application provides an adapter oligonucleotide, which comprises a first strand and a second strand. Wherein, the first strand comprises an upstream sequencing primer sequence, an optional adapter spacer sequence, an optional linker spacer sequence and a restriction sequence III from the 5' end to the 3' end, and the second strand comprises the reverse complementary sequence of a downstream sequencing primer sequence, the reverse complementary sequence of the optional adapter spacer sequence, the reverse complementary sequence of the optional linker spacer sequence and a restriction sequence IV from the 3' end to the 5' end.

[0527] Wherein, the linker spacer sequence is the same as the linker spacer sequence described in the forty-ninth aspect.

[0528] Wherein, the restriction sequence III and the restriction sequence IV form the cohesive ends after cleavage of the restriction endonuclease described in the forty-ninth aspect.

[0529] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27.

[0530] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26.

[0531] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform. Further optionally, it is a single-strand sequencing primer or a double-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0532] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform. Further optionally, it is a single-strand sequencing primer or a double-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0533] In some embodiments, the adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence is not reverse complementary.

[0534] In some embodiments, the first strand further comprises an upstream tag sequence, which is located at the 5' end or 3' end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or, the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3' end or 5' end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0535] In some embodiments, the linker spacer sequence and the reverse complementary sequence of the linker spacer sequence are at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides in length.

[0536] The fifty-second aspect of the present application provides a linker oligonucleotide comprising a first strand and a second strand, wherein the first strand comprises, from the 5'-end to the 3'-end, an upstream sequencing solid-phase binding sequence, an upstream sequencing primer sequence, an optional linker spacer sequence, an optional adapter spacer sequence, and a restriction sequence III, and the second strand comprises, from the 3'-end to the 5'-end, the reverse complementary sequence of the downstream sequencing solid-phase binding sequence, the reverse complementary sequence of the downstream sequencing primer sequence, the reverse complementary sequence of the optional linker spacer sequence, the reverse complementary sequence of the optional adapter spacer sequence, and a restriction sequence IV.

[0537] wherein the adapter spacer sequence is the same as the adapter spacer sequence described in the forty-ninth aspect.

[0538] wherein the restriction sequence III and the restriction sequence IV constitute the sticky ends after cleavage by the restriction endonuclease described in the forty-ninth aspect.

[0539] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform, and further optionally, it is P5 or P7 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.24 or 25.

[0540] In some embodiments, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform, and further optionally, it is P7 or P5 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.25 or 24.

[0541] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and further optionally, it is read1 or read2 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.26 or 27.

[0542] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and further optionally, it is read2 or read1 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.27 or 26.

[0543] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0544] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0545] In some embodiments, the upstream sequencing primer sequence is the sequencing primer sequence of the MGI platform. Further optionally, it is the first-strand sequencing primer or the second-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0546] In some embodiments, the downstream sequencing primer sequence is the sequencing primer sequence of the MGI platform. Further optionally, it is the first-strand sequencing primer or the second-strand sequencing primer of the MGI platform. Further optionally, it is Read1 or Read2 of the MGI platform.

[0547] In some embodiments, the adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence is not reverse complementary, and the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence is not reverse complementary.

[0548] In some embodiments, the first strand further comprises an upstream tag sequence, which is located at the 5' end or the 3' end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3' end or the 5' end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0549] In some embodiments, the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0550] The fifty-third aspect of the present application provides a method for constructing a sequencing library, which includes: (1) using a transposase complex embedded with the adapter oligonucleotide according to the forty-ninth aspect to fragment the target sequence; (2) using a DNA polymerase, the restriction endonuclease according to the forty-ninth aspect, and a ligase for treatment. Optionally, adding the adapter oligonucleotide according to the fiftieth aspect and performing denaturation and annealing before the ligase treatment; and (3) optionally, performing PCR using an extension primer pair,

[0551] wherein, the upstream primer of the extension primer pair includes an upstream sequencing solid-phase binding sequence and the upstream sequencing primer sequence from the 5'-end to the 3'-end, and the downstream primer of the extension primer pair includes a downstream sequencing solid-phase binding sequence and the downstream sequencing primer sequence from the 5'-end to the 3'-end.

[0552] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25.

[0553] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24.

[0554] In some embodiments, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0555] In some embodiments, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the MGI platform. Further optionally, it is the upstream splint adapter sequence or the downstream splint adapter sequence of the MGI platform. Further optionally, it is the linear sequence of the vesicle adapter or the vesicular sequence of the vesicle adapter of the MGI platform.

[0556] In some embodiments, the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence does not reverse complement when present.

[0557] In some embodiments, the upstream primer further comprises an upstream tag sequence, which is located at the 5' end or 3' end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the downstream primer further comprises a downstream tag sequence, which is located at the 5' end or 3' end of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0558] A method for constructing a sequencing library according to the fifty-fourth aspect of the present application, which comprises: (1) using a transposase complex embedded with the adapter oligonucleotide according to the forty-ninth aspect to fragment the target sequence; (2) using a DNA polymerase, the restriction endonuclease according to the forty-ninth aspect and a ligase for treatment, wherein the adapter oligonucleotide according to the fifty-first aspect is added before the ligase treatment; and (3) performing PCR using an extension primer pair.

[0559] Wherein, the upstream primer of the extension primer pair comprises an upstream sequencing solid-phase binding sequence and the upstream sequencing primer sequence from the 5' end to the 3' end, and the downstream primer of the extension primer pair comprises a downstream sequencing solid-phase binding sequence and the downstream sequencing primer sequence from the 5' end to the 3' end.

[0560] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P5 or P7 of the Illumina platform, and further optionally, it is shown as SEQ ID NO.24 or 25.

[0561] In some embodiments, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P7 or P5 of the Illumina platform, and further optionally, it is shown as SEQ ID NO.25 or 24.

[0562] In some embodiments, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the MGI platform, further optionally, it is an upstream splint adapter sequence or a downstream splint adapter sequence of the MGI platform, and further optionally, it is a linear sequence of the vesicle adapter or a vesicular sequence of the vesicle adapter of the MGI platform.

[0563] In some embodiments, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the MGI platform, further optionally, it is an upstream splint adapter sequence or a downstream splint adapter sequence of the MGI platform, and further optionally, it is a linear sequence of the vesicle adapter or a vesicular sequence of the vesicle adapter of the MGI platform.

[0564] In some embodiments, when present, the reverse complementary sequence of the upstream sequencing solid-binding sequence and the downstream sequencing solid-binding sequence is not reverse complementary.

[0565] In some embodiments, the upstream primer further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the downstream primer further comprises a downstream tag sequence, which is located at the 5'-end or 3'-end of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0566] The fifty-fifth aspect of the present application provides a method for constructing a sequencing library, which includes: (1) using a transposase complex encapsulated with the adapter oligonucleotide according to the forty-ninth aspect to fragment the target sequence; and (2) treating with a DNA polymerase, the restriction endonuclease according to the forty-ninth aspect and a ligase, wherein the adapter oligonucleotide according to the fifty-second aspect is added before the ligase treatment.

[0567] The fifty-sixth aspect of the present application provides a kit, which comprises

[0568] (1) the adapter oligonucleotide according to the forty-ninth aspect, the adapter oligonucleotide according to the fifty-first aspect and an extension primer pair; or

[0569] (2) the adapter oligonucleotide according to the forty-ninth aspect and the adapter oligonucleotide according to the fifty-second aspect,

[0570] wherein the upstream primer of the extension primer pair comprises an upstream sequencing solid-binding sequence and the upstream sequencing primer sequence from the 5'-end to the 3'-end, and the downstream primer of the extension primer pair comprises a downstream sequencing solid-binding sequence and the downstream sequencing primer sequence from the 5'-end to the 3'-end.

[0571] In some embodiments, the upstream primer further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the downstream primer further comprises a downstream tag sequence, which is located at the 5'-end or 3'-end of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

[0572] In some embodiments, the kit further comprises one or more of the following: (1) a transposase; (2) a DNA polymerase; (3) a restriction endonuclease as described in the forty-ninth aspect; and (4) a ligase.

[0573] In some embodiments, the DNA polymerase is any conventional DNA polymerase known to those of ordinary skill in the art that can be used for PCR, including but not limited to Taq DNA polymerase, DNA polymerases I-V, Tth DNA polymerase, Pfu DNA polymerase, Escherichia coli (E. coli) DNA polymerase I, Bst DNA polymerase, T7 DNA polymerase, Vent DNA polymerase, and KOD DNA polymerase. BRIEF DESCRIPTION OF THE DRAWINGS

[0574] Figure 1 : Principle of library construction by the fragmentation enzyme method and the principle of library construction by the conventional transposase method;

[0575] Figure 2 : Schematic diagram of the structure of the Tn5-Y complex;

[0576] Figure 3 : Schematic diagram of the structure of the Tn5-Y-U complex;

[0577] Figure 4 : Schematic diagram of the structure of the Tn5-oligo complex;

[0578] Figure 5 : Schematic diagram of the structure of the Tn5-oligo-PCR-free complex;

[0579] Figure 6 : Schematic diagram of the structure of the Tn5-oligo-Y complex;

[0580] Figure 7 : Schematic diagram of the structure of the Tn5-CUT complex;

[0581] Figure 8 : Flow chart of library construction of the Tn5-Y complex;

[0582] Figure 9 : Flow chart of library construction of the Tn5-Y-U complex;

[0583] Figure 10 : Library yields obtained by library construction using the fragmentation enzyme method, the conventional transposase method, the Tn5-Y complex, and the Tn5-Y-U complex, respectively;

[0584] Figure 11A: Library peak graphs obtained by constructing libraries using the fragmentation enzyme method, conventional transposase method, Tn5-Y complex, and Tn5-Y-U complex respectively at an input amount of 100 pg;

[0585] Figure 11B : Library peak graphs obtained by constructing libraries using the fragmentation enzyme method, conventional transposase method, Tn5-Y complex, and Tn5-Y-U complex respectively at an input amount of 1 ng;

[0586] Figure 11C : Library peak graphs obtained by constructing libraries using the fragmentation enzyme method, conventional transposase method, Tn5-Y complex, and Tn5-Y-U complex respectively at an input amount of 10 ng;

[0587] Figure 12 : Library construction flow chart of the Tn5-oligo complex;

[0588] Figure 13 : Library construction flow chart of the Tn5-oligo-PCR-free complex;

[0589] Figure 14 : Conventional library construction flow chart of the Tn5-oligo-Y complex;

[0590] Figure 15 : PCR-free library construction flow chart of the Tn5-oligo-Y complex;

[0591] Figure 16 : Conventional library construction flow chart of the Tn5-CUT complex;

[0592] Figure 17 : PCR-free library construction flow chart of the Tn5-CUT complex;

[0593] Figure 18 : Comparison of library yields of conventional transposase library construction, Tn5-oligo complex library construction, conventional library construction of Tn5-oligo-Y complex, and Tn5-CUT complex library construction;

[0594] Figure 19 : Comparison of library peak graphs of conventional transposase library construction, Tn5-oligo complex library construction, conventional library construction of Tn5-oligo-Y complex, and Tn5-CUT complex library construction;

[0595] Figure 20 : Library yields obtained by conventional transposase library construction, Tn5-oligo complex library construction, and library construction using the adapter replacement strategy;

[0596] Figure 21 : Ligation efficiency between oligonucleotide adapters containing different lengths of oligo and linkers;

[0597] Figure 22 : Comparison of library yields of library construction with conventional transposase, library construction with Tn5-Y-U complex, library construction with Tn5-Y-U1 complex, library construction with Tn5-Y-U2 complex, and library construction with Tn5-Y-U3 complex;

[0598] Figure 23 : Comparison of library peak maps of library construction with conventional transposase, library construction with Tn5-Y-U complex, library construction with Tn5-Y-U1 complex, library construction with Tn5-Y-U2 complex, and library construction with Tn5-Y-U3 complex;

[0599] Figure 24 : Comparison of library yields of library construction with conventional transposase, library construction with Tn5-oligo complex, library construction with Tn5-oligo1 complex, and library construction with Tn5-oligo2 complex;

[0600] Figure 25 : Comparison of library peak maps of library construction with conventional transposase, library construction with Tn5-oligo complex, library construction with Tn5-oligo1 complex, and library construction with Tn5-oligo2 complex.

[0601] Specific implementation mode (Example)

[0602] The technical solutions of the present application will be further described below in conjunction with the accompanying drawings and through specific implementation modes. However, the following examples are only simple examples of the present application and do not represent or limit the scope of the protection of the rights of the present application. The scope of protection of the present application shall be subject to the claims.

[0603] In the following examples, unless otherwise specified, the reagents and consumables used are purchased from conventional reagent manufacturers in the art; unless otherwise specified, the experimental methods and technical means used are conventional methods and means in the art.

[0604] Example 1

[0605] Preparation of different Tn5 transposase complexes

[0606] Oligonucleotides Oligo 1 and Oligo 2 were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Oligo1 includes SEQ ID NO.1-6, and Oligo2 includes SEQ ID NO.7-9. The specific sequences are as follows:

[0607] Table 1

[0608]

[0609] Dissolve Oligo 1 and Oligo 2 to 10 μM respectively using PBS. Take 10 μl of Oligo 1 + 10 μl of Oligo 2 to form Reaction 1. Vortex Reaction 1 thoroughly to mix well, and briefly centrifuge to return the solution to the bottom of the tube. Place it in a PCR instrument and perform the following reaction program:

[0610] Table 2

[0611] Heat lid 105℃ 75℃ 15 min 60℃ 10 min 50℃ 10 min 40℃ 10 min 25℃ 25 min

[0612] After the reaction is completed, name the product of Reaction 1 as Tn5 Adapter.

[0613] Subsequently, add the following reaction components in sequence in a sterilized PCR tube:

[0614] Table 3

[0615] Component 2 μg system Tn5 (500 ng / μl) 4 μl Tn5 Adapter 7 μl PBS 39 μl

[0616] Note: Tn5 in Table 2 is obtained by expression from the psfTn5 plasmid (Addgene plasmid #79107).

[0617] Use a pipette to gently pipette 20 times to mix well, and place it at 30 °C for reaction for 1 h. Name the reaction product as Tn5 complex and store it at -30 to -15 °C.

[0618] The sequence of Oligo A is: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6,

[0619] The sequence of Oligo B is: SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9.

[0620] Among them, SEQ ID NO.1 and SEQ ID NO.7 are used to form the Tn5-Y complex, as Figure 2 shown; SEQ ID NO.2 and SEQ ID NO.7 are used to form the Tn5-Y-U complex, as Figure 3 shown; SEQ ID NO.8 and SEQ ID NO.3 are used to form the Tn5-oligo complex, as Figure 4 shown; SEQ ID NO.8 and SEQ ID NO.4 are used to form the Tn5-oligo-PCR-free complex, as Figure 5 shown; SEQ ID NO.8 and SEQ ID NO.5 are used to form the Tn5-oligo-Y complex, as Figure 6as shown; SEQ ID NO.6 and SEQ ID NO.9 are used to form the Tn5-CUT complex, as Figure 7 shown.

[0621] Example 2

[0622] In this example, the 293 cell gDNA genome was used as an exemplary template, and experiments were conducted using the methods of "conventional Tn5 product library construction", "fragmentase method library construction", "Tn5-Y complex library construction", and "Tn5-Y-U complex library construction" for comparison to illustrate the method steps and beneficial effects of the present application.

[0623] 1. Conventional Tn5 product library construction (using Vazyme product TD502)

[0624] 100 pg, 1 ng, and 10 ng of purified 293 cell gDNA were respectively used as the starting DNA templates, and library preparation was carried out according to the "09 / Experimental Procedure" in the Vazyme #TD502 product manual.

[0625] Next-generation sequencing

[0626] The library was sent to Nanjing Shihe Gene Biotechnology Co., Ltd. for sequencing, and the sequencing instrument was HiseqX.

[0627] 2. Fragmentase method library construction (using Vazyme product ND617)

[0628] 100 pg, 1 ng, and 10 ng of purified 293 cell gDNA were respectively used as the starting DNA templates, and library preparation was carried out according to the "08 / Standard Experimental Protocol" in the Vazyme #ND617 product manual.

[0629] Next-generation sequencing

[0630] Same as the next-generation sequencing method in the above conventional Tn5 product library construction method.

[0631] 3. Tn5-Y complex library construction (using some reagents in Vazyme product TD502)

[0632] DNA fragmentation

[0633] Thaw 5×TTBL (Vazyme #TD502) at room temperature, invert it up and down to mix well and set aside. Confirm whether 5×TS (Vazyme #TD502) is at room temperature, and gently flick the tube wall to check for precipitation. If there is precipitation, heat it at 37°C and vortex to mix well, and the precipitation can be dissolved.

[0634] Prepare the following reaction system in a sterilized PCR tube, where the gDNA is 100 pg, 1 ng, and 10 ng of purified 293 cell gDNA respectively, as the starting DNA template:

[0635] Table 4

[0636] Component Volume 5×TTBL 4 μl gDNA 1 μl Tn5-Y complex 1 μl <![CDATA[ddH2O]]> To 20 μl

[0637] Use a pipette to gently pipette up and down 20 times to mix well.

[0638] Place the reaction tube in a PCR instrument and run the following reaction program:

[0639] Table 5

[0640] Heat lid 105℃ 55℃ 10 min 10℃ Hold

[0641] DNA fragment repair and ligation

[0642] Immediately after the reaction, add 5 μl of 5×TS to the product, use a pipette to gently pipette up and down to mix well, place it at room temperature for 5 min, and then immediately prepare the repair and ligation system and carry out the reaction. The specific system and reaction are as follows:

[0643] Table 6

[0644] Component Volume Fragmentation product 25 μl Klenow fragment (NEB#M0212V) 1 μl dNTP 2 μl T4 DNA ligase (Vazyme#C301) 1 μl ATP (10 mM) (Sigma#11140965001) 1 μl Pure water 15 μl NEBuffer2 (NEB#B7002S) 5 μl

[0645] NEBuffer 2: 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.9, 25°C, the same below.

[0646] After pipetting and mixing, carry out the following reaction:

[0647] Table 7

[0648] Heat lid 105℃ 37℃ 30 min 16℃ 30 min 72℃ 20 min 4℃ Hold

[0649] Purification of the repair and ligation product

[0650] Transfer 60 μl of magnetic beads (Nanjing Novoprotein product N411) into the above PCR reaction product, vortex or pipette 10 times to ensure the whole system is homogeneous, and incubate at room temperature for 5 min. Briefly centrifuge the reaction tube and place it on a magnetic stand to separate the magnetic beads and the liquid. After the solution becomes clear (about 5 min), carefully remove the supernatant, taking care not to disturb the magnetic beads. Keep the centrifuge tube on the magnetic stand all the time, add 200 μl of freshly prepared 80% ethanol to wash the magnetic beads, incubate at room temperature for 30 sec, and carefully remove the supernatant. Repeat this step for a total of two washes. Keep the centrifuge tube on the magnetic stand all the time, open the lid and air-dry for 3 - 5 min. After the magnetic beads are dried, take the centrifuge tube off the magnetic stand, add 22 μl of sterilized ultrapure water for elution, vortex or pipette 10 times to fully mix the magnetic beads, and incubate at room temperature for 5 min. Briefly centrifuge the PCR tube to collect and place it on the magnetic stand to separate the magnetic beads and the liquid. After the solution becomes clear (about 5 min), carefully pipette 20 μl of the supernatant and transfer it to a new EP tube, and store it at -30 to -15 °C.

[0651] PCR Enrichment

[0652] Immediately after the reaction is completed, add 5 μl of 5×TS (Vazyme #TD502) to the product, gently pipette to mix well, place it at room temperature for 5 min, then immediately prepare the PCR system and perform the PCR reaction. The specific system and reaction are as follows:

[0653] Table 8

[0654] Component Volume Fragmentation product 25 μl 5×TAB (Vazyme#TD502) 10 μl N5XX* 5 μl N7XX* 5 μl TAE 1 μl Pure water 4 μl

[0655] *8 kinds of N5XX and 12 kinds of N7XX are from Nanjing Novoprotein product TD202.

[0656] After the above system is pipetted and mixed well, perform the following reaction:

[0657] Table 9

[0658]

[0659] PCR Product Purification

[0660] Vortex and mix the vortex oscillation DNA purification magnetic beads (Nanjing Novoprotein product N411), and pipette 25 μl of the magnetic beads into the above PCR reaction product. Vortex or pipette 10 times to ensure the uniformity of the whole system, and incubate at room temperature for 5 min. Briefly centrifuge the reaction tube and place it on a magnetic stand to separate the magnetic beads and the liquid. After the solution becomes clear (about 5 min), carefully transfer the supernatant to a new centrifuge tube, taking care not to disturb the magnetic beads. Pipette 7.5 μl of the magnetic beads into the above supernatant, vortex or pipette 10 times to ensure the uniformity of the whole system, and incubate at room temperature for 5 min. Place it on the magnetic stand until the solution becomes clear, and remove the supernatant. Keep the centrifuge tube on the magnetic stand all the time, add 200 μl of freshly prepared 80% ethanol to wash the magnetic beads, incubate at room temperature for 30 sec, and carefully remove the supernatant. Repeat this step for a total of two washes. Keep the centrifuge tube on the magnetic stand all the time, open the lid and air-dry for 3 - 5 min. After the magnetic beads are dried, remove the centrifuge tube from the magnetic stand, add 22 μl of sterile ultrapure water for elution, vortex or pipette 10 times to fully mix the magnetic beads, and incubate at room temperature for 5 min. Briefly centrifuge the PCR tube to collect it and place it on the magnetic stand to separate the magnetic beads and the liquid. After the solution becomes clear (about 5 min), carefully pipette 20 μl of the supernatant and transfer it to a new EP tube, and store it at -30 to -15 °C.

[0661] Library quality detection

[0662] Detect the library length distribution of the prepared library on an Agilent 2100 Bioanalyzer.

[0663] Next-generation sequencing

[0664] Same as the above conventional Tn5 product library construction method.

[0665] 4. Library construction of Tn5-Y-U complex (using some reagents in Novoprotein product TD502)

[0666] DNA fragmentation

[0667] Thaw 5×TTBL (Vazyme#TD502) at room temperature, invert it up and down to mix well and set aside. Confirm whether 5×TS (Vazyme#TD502) is at room temperature, and flick the tube wall to check for precipitation. If there is precipitation, heat it at 37 °C and vortex to mix well, and the precipitation can be dissolved.

[0668] Prepare the following reaction system in a sterile PCR tube, where the gDNA is 100 pg, 1 ng, and 10 ng of purified 293 cell gDNA as the starting DNA template respectively:

[0669] Table 10

[0670]

[0671]

[0672] Gently pipette up and down 20 times to mix well.

[0673] Place the reaction tube in a PCR instrument and run the following reaction program:

[0674] Table 11

[0675] Heat lid 105℃ 55℃ 10 min 10℃ Hold

[0676] DNA fragment repair and ligation

[0677] Immediately after the reaction, add 5 μl of 5×TS (Vazyme#TD502) to the product, gently pipette up and down to mix well, place at room temperature for 5 min, and then immediately prepare the repair and ligation system and carry out the reaction. The specific system and reaction are as follows:

[0678] Table 12

[0679] Component Volume Fragmentation product 25 μl Klenow fragment (NEB#M0212V) 1 μl FEN1 (500 ng / μl)* 1 μl dNTP 2 μl T4 DNA ligase (Vazyme#C301) 1 μl ATP (10 mM) (Sigma#11140965001) 1 μl NEBuffer2 5 μl Pure water 14 μl

[0680] *FEN1 was expressed and obtained by the method disclosed in the reference: Bornarth, C.J., Ranalli, T.A., Henricksen, L.A., Wahl, A.F., and Bambara, R.A. (1999) Biochemistry 38, 13347 - 13354.

[0681] After pipetting and mixing, carry out the following reaction:

[0682] Table 13

[0683] Heat lid 105℃ 20℃ 30 min 72℃ 20 min 4℃ Hold

[0684] Purification of the repair and ligation product

[0685] Transfer 60 μl of magnetic beads (Novizan product N411, Nanjing) into the above PCR reaction product, vortex or pipette 10 times to ensure the uniformity of the whole system, and incubate at room temperature for 5 min. Centrifuge the reaction tube briefly and place it on a magnetic stand to separate the magnetic beads and the liquid. After the solution becomes clear (about 5 min), carefully remove the supernatant, taking care not to disturb the magnetic beads. Keep the centrifuge tube on the magnetic stand all the time, add 200 μl of freshly prepared 80% ethanol to wash the magnetic beads, incubate at room temperature for 30 sec, and carefully remove the supernatant. Repeat this step for a total of two washes. Keep the centrifuge tube on the magnetic stand all the time, open the lid and air-dry for 3 - 5 min. After the magnetic beads are dried, take the centrifuge tube off the magnetic stand, add 22 μl of sterilized ultrapure water for elution, vortex or pipette 10 times to fully mix the magnetic beads, and incubate at room temperature for 5 min. Briefly centrifuge the PCR tube to collect it and place it on the magnetic stand to separate the magnetic beads and the liquid. After the solution becomes clear (about 5 min), carefully pipette 20 μl of the supernatant and transfer it to a new EP tube, and store it at -30 to -15 °C.

[0686] The specific steps of PCR enrichment, PCR product purification, library quality detection and second-generation sequencing are the same as the above Tn5-Y complex library construction method.

[0687] The library output is organized as shown in Figure 10 The library peak diagrams are respectively as shown in Figure 11A 、 Figure 11B and Figure 11C The sequencing analysis is shown in Table 14 below.

[0688] Table 14

[0689]

[0690]

[0691] The experimental results show that

[0692] As shown in Figure 10 , when constructing libraries with different starting amounts, the library yields of both Tn5-Y and Tn5-Y-U complexes are much better than those of the libraries prepared by conventional Tn5. Among them, the library yield of the Tn5-Y-U complex exceeds that of the Tn5-Y complex at each input amount, indicating that adding U base modification to the Y-type adapter embedded with transposase can overcome the library loss caused by polymerase strand displacement activity.

[0693] As shown in Figure 11A 、 Figure 11B and Figure 11C , the library distribution shows that at different input amounts, the fragment size distributions of the Tn5-Y and Tn5-Y-U complexes are the same as those of the conventional Tn5 library, and both can effectively fragment the genome.

[0694] The sequencing analysis results are shown in Table 14. The Uniformity value shows that Tn5-Y-U improves the library uniformity by about twice compared with conventional Tn5 and significantly reduces the requirement for sequencing depth (requiring a depth of more than 10X). Uniformity refers to the degree of uniform distribution of the read data in the genome or target region, and the smaller the value, the better the uniformity. There are no differences in conventional mapping rate (the proportion of read data that can be mapped to the reference genome), coverage (the degree of coverage on the reference genome after the read data is assembled, the higher the better), and softclip (a read matches different regions of the reference genome), etc.

[0695] Example 3

[0696] In this example, 10 ng of 293 cell gDNA was used as the DNA template, and the following were used respectively:

[0697] ① Construct a library using a conventional Tn5 product;

[0698] ② The library construction method of the Tn5-oligo complex (the process is as Figure 12 shown). The oligo region in the Tn5-oligo complex is 12 nt (underlined in SEQ ID NO.3), and the Tn5-oligo-adaptor used for ligation is SEQ ID NO.10;

[0699] ③ The library construction method of the Tn5-oligo-PCR-free complex (the process is as Figure 13 shown). The oligo region in the Tn5-oligo-Y complex is 12 nt (underlined in SEQ ID NO.4), and the Tn5-oligo-PCR-free adaptor used for ligation is SEQ ID NO.11;

[0700] ④: The conventional library construction method of the Tn5-oligo-Y complex (the process is as Figure 14 shown). The oligo region in the Tn5-oligo-Y complex is 6 nt (underlined in SEQ ID NO.5), and the Tn5-oligo-Y adaptor used for ligation is annealed from SEQ ID NO.10 and SEQ ID NO.13;

[0701] ⑤: The PCR-free library construction method of the Tn5-oligo-Y complex (the process is as Figure 15As shown in the figure, the oligo region in the Tn5-oligo-Y complex is 6 nt (underlined in SEQ ID NO.5), and the Tn5-oligo-Y-PCR-free adaptor for ligation is annealed from SEQ ID NO.12 and SEQ ID NO.14;

[0702] ⑥: Library construction method for the Tn5-oligo-CUT complex (the process is as Figure 16 shown in the figure). The Tn5-oligo-CUT complex contains a sticky-end recognition region (underlined in SEQ ID NO.6), and the Tn5-oligo adaptor for ligation is annealed from SEQ ID NO.13 and SEQ ID NO.15; (similarly, a PCR-free library construction of the Tn5-CUT complex can also be achieved using an adaptor, and the process is as Figure 17 shown in the figure);

[0703] For experimental comparison, the adaptors involved in the foregoing part are as follows in the table:

[0704] Table 15

[0705]

[0706] The specific steps are as follows:

[0707] ①: Library construction for the conventional Tn5 product, and the specific steps are the same as those in Example 2 above.

[0708] ②: Library construction for the Tn5-oligo complex (using some reagents in Vazyme#TD502)

[0709] DNA fragmentation

[0710] Thaw 5×TTBL (Vazyme#TD502) at room temperature, invert it up and down to mix well and set aside. Confirm whether 5×TS (Vazyme#TD502) is at room temperature, and flick the tube wall gently to check for precipitation. If there is precipitation, heat it at 37°C and vortex it to mix well, and the precipitation can be dissolved.

[0711] Prepare the following reaction system in a sterilized PCR tube:

[0712] Table 16

[0713] Component Volume 5×TTBL 4 μl gDNA (10 ng) 1 μl Tn5-oligo complex 1 μl Pure water To 20 μl

[0714] Use a pipette to gently pipette 20 times to mix well.

[0715] Place the reaction tube in a PCR instrument and run the following reaction program:

[0716] Table 17

[0717] Heat lid 105℃ 55℃ 10 min 10℃ Hold

[0718] DNA fragment repair and ligation

[0719] Dilute Sequence 8 to 10 μM with sterile water for later use. Immediately after the reaction is completed, add 5 μl of 5×TS to the product, gently pipette and mix well, place at room temperature for 5 min, and then immediately prepare the repair and ligation system and carry out the reaction. The specific system and reaction are as follows:

[0720] Table 18

[0721]

[0722] After pipetting and mixing well, carry out the following reaction:

[0723] Table 19

[0724] Heat lid 105℃ 20℃ 30 min 4℃ Hold

[0725] The specific steps for purifying the repair and ligation product, enriching by PCR, purifying the PCR product, detecting the library quality, and second-generation sequencing are the same as those in Example 2.

[0726] ③: Construction of Tn5-oligo-PCR-free complex library (using some reagents in Vazyme#TD502)

[0727] This protocol does not require a PCR enrichment step. For DNA fragmentation, use the Tn5-oligo-PCR-free complex prepared in Example 1. For DNA fragment repair and ligation, use Tn5-oligo-PCR-free adaptor (SEQ ID NO.11). The remaining steps are the same as those in ② above for constructing the Tn5-oligo complex library.

[0728] ④: Conventional method for constructing Tn5-oligo-Y complex library (using some reagents in Vazyme#TD502)

[0729] For DNA fragmentation, use the Tn5-oligo-Y complex prepared in Example 1. For DNA fragment repair and ligation, use the Tn5-oligo-Y adaptor, which is annealed from SEQ ID NO.10 and SEQ ID NO.13. The specific steps are as follows:

[0730] Dilute Sequence SEQ ID NO.10 and Sequence SEQ ID NO.13 with sterile water to 10 μM for standby. Take 10 μl of Sequence SEQ ID NO.10 and 10 μl of Sequence SEQ ID NO.13, mix them by vortex oscillation until thoroughly mixed, and briefly centrifuge to return the solution to the bottom of the tube. Place it in a PCR instrument and perform the following reaction program: Incubate at 95 °C for 2 min, then cool down to 25 °C at a gradient of 0.2 °C per second. After the reaction ends, obtain the reaction product, namely Tn5-oligo-Y adaptor, and store it on ice for standby.

[0731] The dosage of Tn5-oligo-Y adaptor in the DNA fragment repair and ligation step is 5 μl, and adjust the dosage of pure water to keep the total volume of the reaction system unchanged. The remaining steps are the same as the above-mentioned ② Tn5-oligo complex library construction.

[0732] ⑤: PCR-free library construction method of Tn5-oligo-Y complex (using some reagents in Vazyme#TD502)

[0733] This protocol does not require a PCR enrichment step. DNA fragmentation uses the Tn5-oligo-Y complex prepared in Example 1, and DNA fragment repair and ligation use the Tn5-oligo-Y-PCR-free adaptor, which is annealed from SEQ ID NO.12 and SEQ ID NO.14. The annealing method is the same as above, and the remaining steps are the same as the above-mentioned ④ conventional library construction method of Tn5-oligo-Y complex.

[0734] ⑥: Library construction method of Tn5-oligo-CUT complex (using some reagents in Vazyme#TD502)

[0735] DNA fragmentation

[0736] Thaw 5×TTBL (Vazyme#TD502) at room temperature, mix it by inverting up and down, and set it aside for standby. Confirm whether 5×TS (Vazyme#TD502) is at room temperature, and flick the tube wall gently to check for precipitation. If there is precipitation, heat it at 37 °C and mix it by vortex oscillation, and the precipitation can be dissolved.

[0737] Prepare the following reaction system in a sterile PCR tube:

[0738] Table 20

[0739] Component Volume 5×TTBL 4 μl gDNA 1 μl Tn5-CUT complex 1 μl Pure water To 20 μl

[0740] Use a pipette to gently pipette 20 times until thoroughly mixed.

[0741] Place the reaction tube in a PCR instrument and run the following reaction program:

[0742] Table 21

[0743] Heat lid 105℃ 55℃ 10 min 10℃ Hold

[0744] DNA fragment repair

[0745] Immediately after the fragmentation reaction, add 5 μl of 5×TS to the product, gently pipette and mix well, place at room temperature for 5 min, and then immediately prepare the repair ligation system and carry out the reaction. The specific system and reaction are as follows:

[0746] Table 22

[0747] Component Volume Fragmentation product 25 μl Klenow fragment (NEB#M0212V) 2 μl dNTP 2 μl Pure water 7 μl NEBBuffer2 4 μl

[0748] NEB Buffer 2: 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.9.

[0749] Purification of the repaired product

[0750] Use 50 μl of magnetic beads (product N411 from Nanjing Novoprotein) for purification, and the operation steps are the same as above.

[0751] Restriction digestion

[0752] Prepare the restriction digestion system and carry out the reaction. The specific system and reaction are as follows:

[0753] Table 23

[0754] Component Volume Purification product 20 μl BspEI (NEB#R0540S) 1 μl 10×NEBufferr3.1 (NEB#B6003S) 3 μl Pure water To 30 μl

[0755] 10×NEBuffer r3.1: 1 M NaCl, 500 mM Tris-HCl, 100 mM MgCl2, 1 mg / ml Recombinant Albumin (pH 7.9)

[0756] After pipetting and mixing well, carry out the following reaction:

[0757] Table 24

[0758] Heat lid 105℃ 37℃ 15 min 80℃ 20 min 4℃ Hold

[0759] Ligation of the DNA fragment restriction digestion product

[0760] The Tn5-CUT adaptor is formed by annealing the sequence SEQ ID NO.13 and the sequence SEQ ID NO.15: Dilute the sequence SEQ ID NO.13 and the sequence SEQ ID NO.15 to 10 μM with sterile water for later use. Take 10 μl of the sequence SEQ ID NO.13 and 10 μl of the sequence SEQ ID NO.15, mix them by vortexing thoroughly, and centrifuge briefly to return the solution to the bottom of the tube. Place it in a PCR instrument and perform the following reaction program: Incubate at 95 °C for 2 min, then cool down at a gradient of 0.2 °C per second to 25 °C. After the reaction, the reaction product, i.e., the Tn5-CUT adaptor, is obtained and stored on ice for later use.

[0761] After the restriction enzyme digestion reaction is completed, prepare the repair and ligation system and carry out the reaction. The specific system and reaction are as follows:

[0762] Table 25

[0763] Component Volume Restriction digestion product 25 μl T4 DNA ligase (Vazyme#C301) 2 μl ATP (10 mM) (Sigma#11140965001) 1 μl Tn5-CUT-adapter 5 μl Pure water 12 μl NEBuffer2 5 μl

[0764] After pipetting and mixing evenly, carry out the following reaction:

[0765] Table 26

[0766] Heat cover 105℃ 20℃ 30min 4℃ Hold

[0767] Purification of the ligation product

[0768] Use 60 μl of magnetic beads (product N411 of Nanjing Novoprotein) for purification. The specific steps are the same as above.

[0769] The steps of PCR enrichment, PCR product purification, library quality detection, and second-generation sequencing are the same as above.

[0770] Statistical library output is as Figure 18 shown, and the library peak Figure 19 As shown in the figure, for the library construction of the Tn5-oligo-PCR-free complex and the Tn5-oligo-Y-PCR-free complex, since no PCR amplification is required, all the products need to be sent for sequencing, and the library output and library peak figure are not statistically analyzed. All the sequencing data are shown in Table 27.

[0771] Table 27

[0772]

[0773]

[0774] Experimental result description

[0775] As Figure 18, The library yields of library construction with the Tn5-oligo complex, conventional library construction with the Tn5-oligo-Y complex, and library construction with the Tn5-CUT complex all have significant advantages compared with conventional Tn5 library construction. Specifically, the library yields of the three test groups are increased by more than about three times compared with the conventional Tn5 library construction group.

[0776] As Figure 19 , the library distribution shows that the library construction with the Tn5-oligo complex, the conventional library construction with the Tn5-oligo-Y complex, and the library construction with the Tn5-CUT complex have the same fragment size distribution as the conventional Tn5 library, and can all effectively fragment the genome.

[0777] The results of sequencing analysis are shown in Table 27. Among them, the Uniformity value shows that the library construction with the Tn5-oligo complex, the library construction with the Tn5-oligo-PCR-free complex, the conventional library construction with the Tn5-oligo-Y complex, the PCR-free library construction with the Tn5-oligo-Y complex, and the library construction with the Tn5-CUT complex improve the library uniformity compared with the conventional Tn5 library construction, and significantly reduce the requirement for sequencing depth (requiring a depth of more than 10X). There is no difference in the mapping rate, coverage, and softclip indicators between each test group and the conventional Tn5 library construction.

[0778] Example 4

[0779] In this example, the gDNA genome of 293 cells is used as an exemplary template, and experiments are conducted and compared using the "conventional Tn5 product library construction method", the "library construction method with the Tn5-oligo complex", and the "adapter replacement strategy disclosed in Chinese Patent CN115552035A" to illustrate the method steps and beneficial effects of the present application.

[0780] 1. The conventional Tn5 product library construction method is the same as that in Example 2.

[0781] 2. The library construction method with the Tn5-oligo complex is the same as that in Example 3.

[0782] 3. The adapter replacement strategy disclosed in Chinese Patent CN115552035A is carried out according to the steps in the aforementioned patent specification.

[0783] Statistical library yields are as Figure 20As shown, the yield of the Tn5-oligo complex library is better than that of the adapter replacement strategy, and both are better than the library yield prepared by conventional Tn5. The adapter replacement strategy theoretically avoids the library loss caused by the uncontrollability of the above-mentioned strand displacement process. However, the overall library construction process involves multiple operations of opening the lid to add reagents and mixing, which is complex, time-consuming, and results in more library output loss. The method of the present application can avoid the above deficiencies.

[0784] Example 5

[0785] On the basis of the aforementioned Tn5-oligo-Y complex, the lengths of the oligos are further set to 0 nt (blunt-end ligation), 1 nt (TA ligation), 2 nt, 3 nt, 4 nt, 5 nt, and 6 nt respectively (that is, the oligo sequence in the original SEQ ID NO.5 is sequentially reduced from 6 nt to 0 nt), and the lengths of the complementary sequences in the adaptor are adjusted accordingly. The ligation efficiency between the adaptors containing oligos of different lengths and the adaptor is tested. The input amounts of T4 ligase (Vazyme#C301) are 5 pmol, 18 pmol, and 360 pmol respectively. The test results are as Figure 21 shown.

[0786] The results show that: under different input amounts of T4 ligase, the oligo length needs to reach at least 3 nt to achieve a good ligation effect. When the input amount of T4 ligase is 360 pmol, the oligos of 3 - 6 nt can all achieve a ligation efficiency of more than 95%.

[0787] Example 6

[0788] On the basis of the Tn5-Y-U complex library construction in Example 2 above, the position of the U base is adjusted, or multiple positions are set to be replaced with U bases. Specifically, multiple forward insertion sequences of Y-type adaptors containing different U bases as shown in the following table are set, and annealed with SEQ ID NO.7 to form Tn5-Y-U1, Tn5-Y-U2, and Tn5-Y-U3 complexes respectively.

[0789] Table 28

[0790] Tn5-Y-U1 SEQ ID NO.16 TCGTCGGCAGCGTCAGATGTGUATAAGAGACAG Tn5-Y-U2 SEQ ID NO.17 TCGTCGGCAGCGTCAGATGUGTATAAGAGACAG Tn5-Y-U3 SEQ ID NO.18 TCGTCGGCAGCGTCAGATGUGTAUAAGAGACAG

[0791] 10 ng of gDNA is input and library construction is carried out according to the process of Example 2, and the library output is as Figure 22 shown, and the library peak type 23 is as shown in the figure.

[0792] As Figure 22 , replacing a single U base at different positions of the ME sequence has no effect on the library output, but the output is significantly reduced after replacing two U bases; as Figure 23When replacing one or more U bases, there is no obvious change in the library peak pattern.

[0793] Example 7

[0794] On the basis of the library construction of the Tn5-oligo complex in Example 3 above, the number of U bases is further set to 2 and 3 to terminate the polymerization reaction. Specifically, SEQ ID NO.19 with 2 U bases is annealed to SEQ ID NO.8 to form the Tn5-oligo1 complex; SEQ ID NO.20 with 3 U bases is annealed to SEQ ID NO.8 to form the Tn5-oligo2 complex. Correspondingly, the Tn5-oligo-linker used for library construction is also replaced with SEQ ID NO.21 and SEQ ID NO.22 with 2 A bases at the ends, and the sequences are as follows:

[0795] Table 29

[0796]

[0797] Input 10 ng of gDNA for library construction, and perform library construction according to the procedure of Example 3. The library output is as Figure 24 shown, and the library peak pattern is as Figure 25 shown.

[0798] As Figure 24 and Figure 25 show, as the number of U bases increases, the library output decreases significantly. The library can be normally fragmented, and there is no obvious change in the peak position.

[0799] Other sequences involved in this application are shown in the following table:

[0800]

[0801]

Claims

1. An adapter oligonucleotide comprising a first strand and a second strand, wherein, The first strand contains, from the 5'-end to the 3'-end, an optional upstream sequencing solid-phase binding sequence, an optional upstream sequencing primer sequence, and a transposase recognition core sequence. The second strand contains, from the 3'-end to the 5'-end, the reverse complementary sequence of an optional downstream sequencing solid-phase binding sequence, the reverse complementary sequence of an optional downstream sequencing primer sequence, and the reverse complementary sequence of the transposase recognition core sequence. Wherein, one or more T bases in the transposase recognition core sequence of the first strand are replaced with damaged bases. Optionally, the 1st or the 1st and 2nd or the 1st, 2nd and 3rd T bases from the 3'-end in the transposase recognition core sequence of the first strand are replaced with damaged bases. Optionally, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and abasic site, preferably U base. The transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase. Further optionally, it is the ME sequence. Further optionally, it is as shown in SEQ ID NO.

23. Optionally, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25. Optionally, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24. Optionally, the upstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27. Optionally, the downstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26. The adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the upstream sequencing primer sequence and the reverse complementary sequence of the downstream sequencing primer sequence do not reverse-complement when present, and the upstream sequencing solid-phase binding sequence and the reverse complementary sequence of the downstream sequencing solid-phase binding sequence do not reverse-complement when present. Optionally, the first strand further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3'-end or 5'-end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

2. An adapter oligonucleotide comprising a first strand and a second strand, wherein, The first strand comprises a transposase recognition core sequence, and the second strand comprises the reverse complementary sequence of the transposase recognition core sequence. Wherein, one or more T bases in the transposase recognition core sequence of the first strand are replaced with damaged bases. Optionally, the first or the first and the second or the first, the second and the third T base(s) counting from the 3'-end in the transposase recognition core sequence of the first strand are replaced with damaged bases. Optionally, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base and abasic site, preferably U base. The transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase. Further optionally, it is an ME sequence. Further optionally, it is as shown in SEQ ID NO.

23.

3. A linker oligonucleotide comprising a first strand and a second strand, wherein, The first strand comprises an upstream sequencing primer sequence and a transposase recognition core sequence from the 5'-end to the 3'-end, and the second strand comprises the reverse complementary sequence of an optionally downstream sequencing primer sequence and the reverse complementary sequence of the transposase recognition core sequence from the 3'-end to the 5'-end. Wherein, one or more T bases in the transposase recognition core sequence of the first strand are replaced with damaged bases. Optionally, the first or the first and the second or the first, the second and the third T base(s) counting from the 3'-end in the transposase recognition core sequence of the first strand are replaced with damaged bases. Optionally, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base and abasic site, preferably U base. The transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase. Further optionally, it is an ME sequence. Further optionally, it is as shown in SEQ ID NO.

23. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27. Optionally, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26. The adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence does not reverse complement when present. Optionally, the first strand further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3'-end or 5'-end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

4. An adapter oligonucleotide comprising a first strand and a second strand, wherein, The first strand comprises an upstream sequencing primer sequence and a transposase recognition core sequence from the 5'-end to the 3'-end, and the second strand comprises the reverse complementary sequence of the downstream sequencing primer sequence and the reverse complementary sequence of the transposase recognition core sequence from the 3'-end to the 5'-end. Wherein, one or more T bases in the transposase recognition core sequence of the first strand are replaced with damaged bases. Optionally, the first or the first and second or the first, second and third T bases from the 3'-end in the transposase recognition core sequence of the first strand are replaced with damaged bases. Optionally, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base and abasic site, preferably U base. The transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase. Further optionally, it is the ME sequence. Further optionally, it is as shown in SEQ ID NO.

23. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27. Optionally, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26. The adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the upstream sequencing primer sequence and the reverse complementary sequence of the downstream sequencing primer sequence do not reverse complement. Optionally, the first strand further comprises an upstream tag sequence, which is located at the 5' end or 3' end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3' end or 5' end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

5. An adapter oligonucleotide comprising a first strand and a second strand, wherein, The first strand comprises an upstream sequencing primer sequence and a transposase recognition core sequence from the 5' end to the 3' end, and the second strand comprises the reverse complementary sequence of the transposase recognition core sequence. Wherein, one or more T bases in the transposase recognition core sequence of the first strand are replaced with damaged bases. Optionally, the first or the first and the second or the first, the second and the third T base(s) from the 3' end in the transposase recognition core sequence of the first strand are replaced with damaged bases. Optionally, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base and abasic site, preferably U base. The transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase. Further optionally, it is the ME sequence. Further optionally, it is as shown in SEQ ID NO.

23. Optionally, the upstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27. Optionally, the first strand further comprises an upstream tag sequence, which is located at the 5' end or 3' end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

6. An adapter oligonucleotide comprising a first strand and a second strand, wherein, The first strand comprises an upstream sequencing solid-phase binding sequence, an upstream sequencing primer sequence and a transposase recognition core sequence from the 5' end to the 3' end, and the second strand comprises, from the 3' end to the 5' end, the reverse complementary sequence of an optional downstream sequencing solid-phase binding sequence, the reverse complementary sequence of an optional downstream sequencing primer sequence and the reverse complementary sequence of the transposase recognition core sequence. Wherein, one or more T bases in the transposase recognition core sequence of the first strand are replaced with damaged bases. Optionally, the first or the first and the second or the first, the second and the third T base(s) from the 3' end in the transposase recognition core sequence of the first strand are replaced with damaged bases. Optionally, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base and abasic site, preferably U base. The transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase. Further optionally, it is the ME sequence. Further optionally, it is as shown in SEQ ID NO.

23. Optionally, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25. Optionally, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24. Optionally, the upstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27. Optionally, the downstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26. The adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the reverse complementary sequence of the downstream sequencing primer sequence do not reverse complement when present, and the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the reverse complementary sequence of the downstream sequencing solid-phase binding sequence do not reverse complement when present. Optionally, the first strand further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3'-end or 5'-end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

7. An adapter oligonucleotide comprising a first strand and a second strand, wherein, The first strand contains the upstream sequencing solid-phase binding sequence, the upstream sequencing primer sequence, and the transposase recognition core sequence from the 5'-end to the 3'-end, and the second strand contains the reverse complementary sequence of the downstream sequencing solid-phase binding sequence, the reverse complementary sequence of the downstream sequencing primer sequence, and the reverse complementary sequence of the transposase recognition core sequence from the 3'-end to the 5'-end. Wherein, one or more T bases in the transposase recognition core sequence of the first strand are replaced with damaged bases. Optionally, the 1st or the 1st and 2nd or the 1st, 2nd and 3rd T bases from the 3'-end in the transposase recognition core sequence of the first strand are replaced with damaged bases. Optionally, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and abasic site, preferably U base. The transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase. Further optionally, it is the ME sequence. Further optionally, it is as shown in SEQ ID NO.

23. Optionally, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25. Optionally, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24. Optionally, the upstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27. Optionally, the downstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26. The adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary, and the reverse complementary sequences of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence are not reverse complementary. Optionally, the first strand further comprises an upstream tag sequence, which is located at the 5' end or 3' end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3' end or 5' end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

8. An adapter oligonucleotide comprising a first strand and a second strand, wherein, The first strand contains the upstream sequencing solid-phase binding sequence, the upstream sequencing primer sequence, and the transposase recognition core sequence from the 5' end to the 3' end, and the second strand contains the reverse complementary sequence of the transposase recognition core sequence. Wherein, one or more T bases in the transposase recognition core sequence of the first strand are replaced with damaged bases. Optionally, the 1st or the 1st and 2nd or the 1st, 2nd, and 3rd T bases from the 3' end in the transposase recognition core sequence of the first strand are replaced with damaged bases. Optionally, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base and abasic site, preferably U base. The transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase. Further optionally, it is the ME sequence. Further optionally, it is as shown in SEQ ID NO.

23. Optionally, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25. Optionally, the upstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27. Optionally, the first strand further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

9. An adaptor oligonucleotide, which comprises an optional first strand and second strand, wherein, The first strand contains, from the 5'-end to the 3'-end, optionally an upstream sequencing solid-phase binding sequence, an upstream sequencing primer sequence and an adaptor spacer sequence. The second strand contains, from the 3'-end to the 5'-end, the reverse complementary sequence of the optionally downstream sequencing solid-phase binding sequence, the reverse complementary sequence of the downstream sequencing primer sequence, the reverse complementary sequence of the optionally adaptor spacer sequence, the 3'-end portion of the reverse complementary sequence of the transposase recognition core sequence and an A base. Wherein, the 3'-end portion of the reverse complementary sequence of the transposase recognition core sequence is reverse complementary to the 5'-end portion of the transposase recognition core sequence of the first strand of the linker oligonucleotide according to claim 1. Wherein, the 5'-end portion of the transposase recognition core sequence refers to the portion of the transposase recognition core sequence at the 5'-end of the damaged base. Optionally, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25. Optionally, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24. Optionally, the upstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27. Optionally, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26. The adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence does not reverse complement when present, and the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence does not reverse complement when present. Optionally, the first strand further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3'-end or 5'-end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. Wherein, the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

10. An adapter oligonucleotide comprising a first strand and a second strand, wherein, The first strand contains, from the 5'-end to the 3'-end, optionally an upstream sequencing solid-phase binding sequence, an upstream sequencing primer sequence, and an adapter spacer sequence. The second strand contains, from the 3'-end to the 5'-end, the reverse complementary sequence of an optionally downstream sequencing solid-phase binding sequence, the reverse complementary sequence of a downstream sequencing primer sequence, the reverse complementary sequence of the adapter spacer sequence, the 3'-end portion of the reverse complementary sequence of the transposase recognition core sequence, and an A base. Wherein, the 3'-end portion of the reverse complementary sequence of the transposase recognition core sequence is reverse complementary to the 5'-end portion of the transposase recognition core sequence of the first strand of the adapter oligonucleotide according to claim 2. Wherein, the 5'-end portion of the transposase recognition core sequence refers to the portion of the transposase recognition core sequence at the 5'-end of the damaged base. Optionally, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25. Optionally, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27. Optionally, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26. The adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence does not reverse complement when present, and the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence does not reverse complement when present. Optionally, the first strand further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3'-end or 5'-end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. Wherein, the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

11. An adaptor oligonucleotide comprising a first strand and a second strand, wherein, The first strand comprises an upstream sequencing primer sequence and an adapter spacer sequence from the 5'-end to the 3'-end, and the second strand comprises the reverse complementary sequence of the downstream sequencing primer sequence, the reverse complementary sequence of the adapter spacer sequence, the 3'-end portion of the reverse complementary sequence of the transposase recognition core sequence and an A base from the 3'-end to the 5'-end. Wherein, the 3'-end portion of the reverse complementary sequence of the transposase recognition core sequence is reverse complementary to the 5'-end portion of the transposase recognition core sequence of the first strand of the adapter oligonucleotide according to claim 2, and the 5'-end portion of the transposase recognition core sequence refers to the portion of the transposase recognition core sequence at the 5'-end of the damaged base. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27. Optionally, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26. The adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the upstream sequencing primer sequence and the reverse complementary sequence of the downstream sequencing primer sequence do not reverse complement. Optionally, the first strand further comprises an upstream tag sequence located at the 5'-end or 3'-end of the upstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence located at the 3'-end or 5'-end of the reverse complementary sequence of the downstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. Wherein, the adaptor spacer sequence and the reverse complementary sequence of the adaptor spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

12. An adaptor oligonucleotide comprising a first strand and a second strand, wherein, The first strand contains an upstream sequencing solid-phase binding sequence, an upstream sequencing primer sequence and an adaptor spacer sequence from the 5'-end to the 3'-end, and the second strand contains the reverse complementary sequence of a downstream sequencing solid-phase binding sequence, the reverse complementary sequence of a downstream sequencing primer sequence, the reverse complementary sequence of the adaptor spacer sequence, the 3'-end portion of the reverse complementary sequence of the transposase recognition core sequence and an A base from the 3'-end to the 5'-end. Wherein, the 3'-end portion of the reverse complementary sequence of the transposase recognition core sequence is reverse complementary to the 5'-end portion of the transposase recognition core sequence of the first strand of the adaptor oligonucleotide according to claim 2, and the 5'-end portion of the transposase recognition core sequence refers to the portion of the transposase recognition core sequence at the 5'-end of the damaged base. Optionally, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P5 or P7 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.24 or 25. Optionally, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P7 or P5 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.25 or 24. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, further optionally, it is read1 or read2 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.26 or 27. Optionally, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, further optionally, it is read2 or read1 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.27 or 26. The adaptor oligonucleotide is a Y-shaped adaptor oligonucleotide, that is, the upstream sequencing primer sequence and the reverse complementary sequence of the downstream sequencing primer sequence are not reverse complementary, and the upstream sequencing solid-phase binding sequence and the reverse complementary sequence of the downstream sequencing solid-phase binding sequence are not reverse complementary. Optionally, the first strand further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the reverse complementary sequence of the downstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. Wherein, the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

13. An adaptor oligonucleotide comprising a second strand, wherein, The second strand comprises, from the 3' end to the 5' end, the reverse complementary sequence of the downstream sequencing primer sequence, the 3' end portion of the reverse complementary sequence of the transposase recognition core sequence, and an A base. Wherein, the 3' end portion of the reverse complementary sequence of the transposase recognition core sequence is reverse complementary to the 5' end portion of the transposase recognition core sequence of the first strand of the adapter oligonucleotide according to claim 5, wherein the 5' end portion of the transposase recognition core sequence refers to the portion of the transposase recognition core sequence at the 5' end of the damaged base. Optionally, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26. The reverse complementary sequence of the downstream sequencing primer sequence is not reverse complementary to the upstream sequencing primer sequence of the adapter oligonucleotide according to claim 5. Optionally, the second strand further comprises the reverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the reverse complementary sequence of the downstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

14. An adaptor oligonucleotide comprising a second strand, wherein, The second strand comprises, from the 3' end to the 5' end, the reverse complementary sequence of the downstream sequencing solid-phase binding sequence, the reverse complementary sequence of the downstream sequencing primer sequence, the 3' end portion of the reverse complementary sequence of the transposase recognition core sequence, and an A base. Wherein, the 3' end portion of the reverse complementary sequence of the transposase recognition core sequence is reverse complementary to the 5' end portion of the transposase recognition core sequence of the first strand of the adapter oligonucleotide according to claim 8, wherein the 5' end portion of the transposase recognition core sequence refers to the portion of the transposase recognition core sequence at the 5' end of the damaged base. Optionally, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24. Optionally, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26. The reverse complementary sequence of the downstream sequencing primer sequence is not reverse complementary to the upstream sequencing primer sequence of the adapter oligonucleotide according to claim 8, and the reverse complementary sequence of the downstream sequencing solid-phase binding sequence is not reverse complementary to the upstream sequencing solid-phase binding sequence of the adapter oligonucleotide according to claim 8. Optionally, the second strand further contains the reverse complementary sequence of the downstream tag sequence, which is located at the 3'-end or 5'-end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

15. A method for constructing a sequencing library, comprising: (1) Use a transposase complex embedded with the adapter oligonucleotide according to claim 1 to fragment the target sequence; (2) Treat with a DNA polymerase intolerant to damaged bases, FEN1 enzyme and ligase. Optionally, add the adapter oligonucleotide according to claim 8 before ligase treatment and perform denaturation and annealing; and (3) Optionally, perform PCR using an extension primer pair. Wherein, the upstream primer of the extension primer pair contains an upstream sequencing solid-phase binding sequence and the upstream sequencing primer sequence from the 5'-end to the 3'-end, and the downstream primer of the extension primer pair contains a downstream sequencing solid-phase binding sequence and the downstream sequencing primer sequence from the 5'-end to the 3'-end. Optionally, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25. Optionally, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24. Optionally, the upstream sequencing solid-phase binding sequence is not reverse complementary to the reverse complementary sequence of the downstream sequencing solid-phase binding sequence when present. Optionally, the upstream primer further contains an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the downstream primer further contains a downstream tag sequence, which is located at the 5'-end or 3'-end of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

16. A method for constructing a sequencing library, comprising: (1) Disrupt the target sequence using a transposase complex encapsulating the adapter oligonucleotide according to claim 2; (2) Treat with a DNA polymerase intolerant to damaged bases, FEN1 enzyme, and ligase, wherein the adapter oligonucleotide according to claim 11 is added and denatured and annealed before ligase treatment; and (3) Perform PCR using extension primer pairs, wherein the upstream primer of the extension primer pair comprises an upstream sequencing solid-phase binding sequence and the upstream sequencing primer sequence from the 5'-end to the 3'-end, and the downstream primer of the extension primer pair comprises a downstream sequencing solid-phase binding sequence and the downstream sequencing primer sequence from the 5'-end to the 3'-end, Optionally, the DNA polymerase intolerant to damaged bases is one or more of DNA polymerases intolerant to U base, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase, and Tgo DNA polymerase, Optionally, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, and Taq DNA ligase, Optionally, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P5 or P7 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.24 or 25, Optionally, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P7 or P5 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.25 or 24, Optionally, when present, the upstream sequencing solid-phase binding sequence and the reverse complementary sequence of the downstream sequencing solid-phase binding sequence are not reverse complementary, Optionally, the upstream primer further comprises an upstream tag sequence, the upstream tag sequence is located at the 5'-end or 3'-end of the upstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or, the downstream primer further comprises a downstream tag sequence, the downstream tag sequence is located at the 5'-end or 3'-end of the downstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

17. A method for constructing a sequencing library, comprising: (1) Disrupt the target sequence using a transposase complex encapsulating the adapter oligonucleotide according to claim 2; and (2) Treat with a DNA polymerase intolerant to damaged bases, FEN1 enzyme, and ligase, wherein the adapter oligonucleotide according to claim 12 is added and denatured and annealed before ligase treatment, Optionally, the DNA polymerase intolerant to damaged bases is one or more of the DNA polymerases intolerant to U base, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase and Tgo DNA polymerase. Optionally, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase and Taq DNA ligase.

18. A method for constructing a sequencing library, comprising: (1) Interrupt the target sequence using the transposase complex embedded with the adapter oligonucleotide according to claim 4; (2) Treat with a DNA polymerase intolerant to damaged bases, FEN1 enzyme and a ligase; and (3) Perform PCR using an extension primer pair. Wherein, the upstream primer of the extension primer pair includes an upstream sequencing solid-phase binding sequence and the upstream sequencing primer sequence from the 5'-end to the 3'-end, and the downstream primer of the extension primer pair includes a downstream sequencing solid-phase binding sequence and the downstream sequencing primer sequence from the 5'-end to the 3'-end. Optionally, the DNA polymerase intolerant to damaged bases is one or more of the DNA polymerases intolerant to U base, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase and Tgo DNA polymerase. Optionally, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase and Taq DNA ligase. Optionally, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25. Optionally, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24. Optionally, the upstream sequencing solid-phase binding sequence and the reverse complementary sequence of the downstream sequencing solid-phase binding sequence do not reverse complement when present. Optionally, the upstream primer further includes an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or, the downstream primer further includes a downstream tag sequence, which is located at the 5'-end or 3'-end of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

19. A method for constructing a sequencing library, comprising: (1) Disrupt the target sequence using a transposase complex encapsulated with the adapter oligonucleotide according to claim 5; (2) Treat with a DNA polymerase intolerant to damaged bases, FEN1 enzyme, and ligase, wherein the adapter oligonucleotide according to claim 13 is added and denatured and annealed before the ligase treatment; and (3) Perform PCR using an extension primer pair, Wherein, the upstream primer of the extension primer pair contains an upstream sequencing solid-phase binding sequence and the upstream sequencing primer sequence from the 5'-end to the 3'-end, and the downstream primer of the extension primer pair contains a downstream sequencing solid-phase binding sequence and the downstream sequencing primer sequence from the 5'-end to the 3'-end, Optionally, the DNA polymerase intolerant to damaged bases is one or more of the DNA polymerases intolerant to U base, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase, and Tgo DNA polymerase, Optionally, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, and Taq DNA ligase, Optionally, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25, Optionally, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24, Optionally, the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence is not reverse complementary when present, Optionally, the upstream primer further contains an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or, the downstream primer further contains a downstream tag sequence, which is located at the 5'-end or 3'-end of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

20. A method for constructing a sequencing library, comprising: (1) Disrupt the target sequence using a transposase complex encapsulated with the adapter oligonucleotide according to claim 7; and (2) Treat with a DNA polymerase intolerant to damaged bases, FEN1 enzyme, and ligase, Optionally, the DNA polymerase intolerant to damaged bases is one or more of the DNA polymerases intolerant to U base, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase, and Tgo DNA polymerase, Optionally, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, and Taq DNA ligase.

21. A method for constructing a sequencing library, comprising: (1) Disrupt the target sequence using a transposase complex embedded with the adapter oligonucleotide according to claim 8; and (2) Treat with a DNA polymerase intolerant of damaged bases, FEN1 enzyme, and ligase, wherein the adapter oligonucleotide according to claim 14 is added and denatured and annealed before the ligase treatment.

22. A kit comprising (1) The adapter oligonucleotide according to claim 2, the adapter oligonucleotide according to claim 11, and an extension primer pair; (2) The adapter oligonucleotide according to claim 2 and the adapter oligonucleotide according to claim 12; (3) The adapter oligonucleotide according to claim 4 and an extension primer pair; (4) The adapter oligonucleotide according to claim 5, the adapter oligonucleotide according to claim 13, and an extension primer pair; (5) The adapter oligonucleotide according to claim 7; or (6) The adapter oligonucleotide according to claim 8 and the adapter oligonucleotide according to claim 14, Among them, The upstream primer of the extension primer pair comprises an upstream sequencing solid-phase binding sequence and the upstream sequencing primer sequence from the 5'-end to the 3'-end, and the downstream primer of the extension primer pair comprises a downstream sequencing solid-phase binding sequence and the downstream sequencing primer sequence from the 5'-end to the 3'-end. Optionally, the upstream primer further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the downstream primer further comprises a downstream tag sequence, which is located at the 5'-end or 3'-end of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. The kit further comprises one or more of the following: (1) Transposase; (2) DNA polymerase intolerant of damaged bases; (3) FEN1 enzyme; and (4) Ligase. Optionally, the DNA polymerase intolerant of damaged bases is one or more of DNA polymerases intolerant of U bases, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase, and Tgo DNA polymerase. Optionally, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, and Taq DNA ligase.

23. An adapter oligonucleotide comprising a first strand and a second strand, wherein, The first strand contains, from the 5'-end to the 3'-end, an optional upstream sequencing solid-phase binding sequence, an optional upstream sequencing primer sequence, a linker spacer sequence, one or more damaged bases, and a transposase recognition core sequence. The second strand contains, from the 3'-end to the 5'-end, the reverse complementary sequence of an optional downstream sequencing solid-phase binding sequence, the reverse complementary sequence of an optional downstream sequencing primer sequence, the reverse complementary sequence of an optional said linker spacer sequence, an optional one or more A bases, and the reverse complementary sequence of the transposase recognition core sequence. The transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase, further optionally, an ME sequence, and further optionally, as shown in SEQ ID NO.

23. Optionally, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform, further optionally, P5 or P7 of the Illumina platform, and further optionally, as shown in SEQ ID NO.24 or 25. Optionally, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform, further optionally, P7 or P5 of the Illumina platform, and further optionally, as shown in SEQ ID NO.25 or 24. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, further optionally, read1 or read2 of the Illumina platform, and further optionally, as shown in SEQ ID NO.26 or 27. Optionally, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, further optionally, read2 or read1 of the Illumina platform, and further optionally, as shown in SEQ ID NO.27 or 26. Optionally, the linker oligonucleotide is a Y-shaped linker oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence do not reverse complement when present, and the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence do not reverse complement when present. Optionally, the first strand further contains an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or, the second strand further contains the reverse complementary sequence of a downstream tag sequence, which is located at the 3'-end or 5'-end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. Optionally, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and abasic site, preferably U base. Among them, the length of the linker spacer sequence and the reverse complementary sequence of the linker spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

24. An adapter oligonucleotide comprising a first strand and a second strand, wherein, The first strand contains a linker spacer sequence, a damaged base, and a transposase recognition core sequence from the 5'-end to the 3'-end. The second strand contains the reverse complementary sequence of the optional linker spacer sequence, an optional A base, and the reverse complementary sequence of the transposase recognition core sequence from the 3'-end to the 5'-end. The transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase. Further optionally, it is the ME sequence. Further optionally, it is as shown in SEQ ID NO.

23. Optionally, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and abasic site, preferably the U base. Among them, the length of the linker spacer sequence and the reverse complementary sequence of the linker spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

25. An adapter oligonucleotide comprising a first strand and a second strand, wherein, The first strand contains a linker spacer sequence, a damaged base, and a transposase recognition core sequence from the 5'-end to the 3'-end. The second strand contains the reverse complementary sequence of the linker spacer sequence, an A base, and the reverse complementary sequence of the transposase recognition core sequence from the 3'-end to the 5'-end. The transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase. Further optionally, it is the ME sequence. Further optionally, it is as shown in SEQ ID NO.

23. Optionally, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and abasic site, preferably the U base. Among them, the length of the linker spacer sequence and the reverse complementary sequence of the linker spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

26. An adapter oligonucleotide, comprising a first strand and a second strand, wherein, The first strand contains a linker spacer sequence, a damaged base, and a transposase recognition core sequence from the 5'-end to the 3'-end. The second strand contains the reverse complementary sequence of the transposase recognition core sequence. The transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase. Further optionally, it is the ME sequence. Further optionally, it is as shown in SEQ ID NO.

23. Optionally, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and abasic site, preferably the U base. Among them, the length of the linker spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

27. An adapter oligonucleotide comprising a first strand and a second strand, wherein, The first strand contains, from the 5'-end to the 3'-end, an upstream sequencing primer sequence, a linker spacer sequence, a damaged base, and a transposase recognition core sequence. The second strand contains, from the 3'-end to the 5'-end, the reverse complementary sequence of an optional downstream sequencing primer sequence, the reverse complementary sequence of an optional linker spacer sequence, an optional A base, and the reverse complementary sequence of the transposase recognition core sequence. The transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase, and further optionally, it is an ME sequence, and further optionally, it is as shown in SEQ ID NO.

23. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and further optionally, it is read1 or read2 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.26 or 27. Optionally, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and further optionally, it is read2 or read1 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.27 or 26. Optionally, the linker oligonucleotide is a Y-shaped linker oligonucleotide, that is, the upstream sequencing primer sequence and the reverse complementary sequence of the downstream sequencing primer sequence, when present, are not reverse complementary. Optionally, the first strand further contains an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further contains the reverse complementary sequence of a downstream tag sequence, which is located at the 3'-end or 5'-end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. Optionally, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and abasic site, and preferably the U base. Wherein, the linker spacer sequence and the reverse complementary sequence of the linker spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

28. An adapter oligonucleotide comprising a first strand and a second strand, wherein, The first strand contains, from the 5'-end to the 3'-end, an upstream sequencing primer sequence, a linker spacer sequence, a damaged base, and a transposase recognition core sequence. The second strand contains, from the 3'-end to the 5'-end, the reverse complementary sequence of the downstream sequencing primer sequence, the reverse complementary sequence of the linker spacer sequence, an A base, and the reverse complementary sequence of the transposase recognition core sequence. The transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase, and further optionally, it is an ME sequence, and further optionally, it is as shown in SEQ ID NO.

23. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence for the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27. Optionally, the downstream sequencing primer sequence is a sequencing primer sequence for the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26. Optionally, the adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence is not reverse complementary. Optionally, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and abasic site, preferably U base. Optionally, the first strand further comprises an upstream tag sequence, which is located at the 5' end or 3' end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3' end or 5' end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. Wherein, the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

29. An adapter oligonucleotide comprising a first strand and a second strand, wherein, The first strand comprises an upstream sequencing primer sequence, an adapter spacer sequence, a damaged base, and a transposase recognition core sequence from the 5' end to the 3' end. The second strand comprises the reverse complementary sequence of the transposase recognition core sequence. The transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase. Further optionally, it is the ME sequence. Further optionally, it is as shown in SEQ ID NO.

23. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence for the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27. Optionally, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and abasic site, preferably U base. Optionally, the first strand further comprises an upstream tag sequence, which is located at the 5' end or 3' end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. Wherein, the adapter spacer sequence has a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

30. An adapter oligonucleotide comprising a first strand and a second strand, wherein, The first strand contains, from the 5'-end to the 3'-end, an upstream sequencing solid-phase binding sequence, an upstream sequencing primer sequence, a linker spacer sequence, a damaged base, and a transposase recognition core sequence. The second strand contains, from the 3'-end to the 5'-end, the reverse complementary sequence of an optional downstream sequencing solid-phase binding sequence, the reverse complementary sequence of an optional downstream sequencing primer sequence, the reverse complementary sequence of an optional said linker spacer sequence, an optional A base, and the reverse complementary sequence of the transposase recognition core sequence. The transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase, and further optionally, it is the ME sequence, and further optionally, it is as shown in SEQ ID NO.

23. Optionally, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform, and further optionally, it is P5 or P7 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.24 or 25. Optionally, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform, and further optionally, it is P7 or P5 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.25 or 24. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and further optionally, it is read1 or read2 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.26 or 27. Optionally, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and further optionally, it is read2 or read1 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.27 or 26. Optionally, the linker oligonucleotide is a Y-shaped linker oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence do not reverse complement when present, and the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence do not reverse complement when present. Optionally, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and abasic site, and preferably the U base. Optionally, the first strand further contains an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or, the second strand further contains the reverse complementary sequence of a downstream tag sequence, which is located at the 3'-end or 5'-end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. Wherein, the length of the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

31. An adapter oligonucleotide comprising a first strand and a second strand, wherein, The first strand contains, from the 5'-end to the 3'-end, an upstream sequencing solid-phase binding sequence, an upstream sequencing primer sequence, an adapter spacer sequence, a damaged base, and a transposase recognition core sequence. The second strand contains, from the 3'-end to the 5'-end, the reverse complementary sequence of the downstream sequencing solid-phase binding sequence, the reverse complementary sequence of the downstream sequencing primer sequence, the reverse complementary sequence of the adapter spacer sequence, an A base, and the reverse complementary sequence of the transposase recognition core sequence. The transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase, and further optionally, it is the ME sequence, and further optionally, as shown in SEQ ID NO.

23. Optionally, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform, and further optionally, it is P5 or P7 of the Illumina platform, and further optionally, as shown in SEQ ID NO.24 or 25. Optionally, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform, and further optionally, it is P7 or P5 of the Illumina platform, and further optionally, as shown in SEQ ID NO.25 or 24. Optionally, the upstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform, and further optionally, it is read1 or read2 of the Illumina platform, and further optionally, as shown in SEQ ID NO.26 or 27. Optionally, the downstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform, and further optionally, it is read2 or read1 of the Illumina platform, and further optionally, as shown in SEQ ID NO.27 or 26. Optionally, the adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary, and the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence are not reverse complementary. Optionally, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and abasic site, and preferably the U base. Optionally, the first strand further contains an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence, and the length is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or, the second strand further contains the reverse complementary sequence of a downstream tag sequence, which is located at the 3'-end or 5'-end of the reverse complementary sequence of the downstream sequencing primer sequence, and the length is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. Wherein, the length of the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

32. An adapter oligonucleotide comprising a first strand and a second strand, wherein, The first strand from the 5'-end to the 3'-end contains an upstream sequencing solid-phase binding sequence, an upstream sequencing primer sequence, an adapter spacer sequence, a damaged base and a transposase recognition core sequence, and the second strand contains the reverse complementary sequence of the transposase recognition core sequence, and the transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase, further optionally, it is an ME sequence, and further optionally, as shown in SEQ ID NO.

23. Optionally, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P5 or P7 of the Illumina platform, and further optionally, as shown in SEQ ID NO.24 or 25. Optionally, the upstream sequencing primer sequence is the sequencing primer sequence of the Illumina platform, further optionally, it is read1 or read2 of the Illumina platform, and further optionally, as shown in SEQ ID NO.26 or 27. Optionally, the damaged base is any one of U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base and abasic site, and preferably the U base. Optionally, the first strand further contains an upstream tag sequence, and the upstream tag sequence is located at the 5'-end or 3'-end of the upstream sequencing primer sequence, and the length is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. Wherein, the length of the adapter spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

33. An adaptor oligonucleotide, which comprises an optional first strand and a second strand, wherein, The first strand from the 5'-end to the 3'-end contains an optional upstream sequencing solid-phase binding sequence, an upstream sequencing primer sequence and an adaptor spacer sequence, and the second strand from the 3'-end to the 5'-end contains the reverse complementary sequence of an optional downstream sequencing solid-phase binding sequence, the reverse complementary sequence of a downstream sequencing primer sequence, the reverse complementary sequence of an optional adaptor spacer sequence, the reverse complementary sequence of an adapter spacer sequence and an A base. Wherein, the reverse complementary sequence of the adapter spacer sequence is reverse complementary to the adapter spacer sequence of the first strand of the adapter oligonucleotide according to claim 23. Optionally, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P5 or P7 of the Illumina platform, and further optionally, as shown in SEQ ID NO.24 or 25. Optionally, the downstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P7 or P5 of the Illumina platform, and further optionally, as shown in SEQ ID NO.25 or 24. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence for the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27. Optionally, the downstream sequencing primer sequence is a sequencing primer sequence for the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26. The adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence do not reverse complement when present, and the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence do not reverse complement when present. Optionally, the first strand further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3'-end or 5'-end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. Wherein, the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

34. An adapter oligonucleotide comprising a first strand and a second strand, wherein, The first strand comprises, from the 5'-end to the 3'-end, optionally an upstream sequencing solid-phase binding sequence, an upstream sequencing primer sequence and an adapter spacer sequence. The second strand comprises, from the 3'-end to the 5'-end, the reverse complementary sequence of an optionally downstream sequencing solid-phase binding sequence, the reverse complementary sequence of a downstream sequencing primer sequence, the reverse complementary sequence of the adapter spacer sequence, the reverse complementary sequence of a linker spacer sequence and an A base, wherein the reverse complementary sequence of the linker spacer sequence is reverse complementary to the linker spacer sequence of the first strand of the linker oligonucleotide according to claim 24. Optionally, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence for the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25. Optionally, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence for the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27. Optionally, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26. The adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence does not reverse complement when present, and the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence does not reverse complement when present. Optionally, the first strand further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3'-end or 5'-end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. Wherein, the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

35. An adapter oligonucleotide comprising a first strand and a second strand, wherein, The first strand contains an upstream sequencing primer sequence and an adapter spacer sequence from the 5'-end to the 3'-end, and the second strand contains the reverse complementary sequence of the downstream sequencing primer sequence, the reverse complementary sequence of the adapter spacer sequence, the reverse complementary sequence of the linker spacer sequence and an A base from the 3'-end to the 5'-end. Wherein, the reverse complementary sequence of the linker spacer sequence is reverse complementary to the linker spacer sequence of the first strand of the linker oligonucleotide according to claim 26. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27. Optionally, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26. The adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence does not reverse complement. Optionally, the first strand further comprises an upstream tag sequence, which is located at the 5' or 3' end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3' or 5' end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. Wherein, the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

36. An adapter oligonucleotide comprising a first strand and a second strand, wherein, The first strand comprises an upstream sequencing solid-phase binding sequence, an upstream sequencing primer sequence and an adapter spacer sequence from the 5' end to the 3' end, and the second strand comprises the reverse complementary sequence of a downstream sequencing solid-phase binding sequence, the reverse complementary sequence of a downstream sequencing primer sequence, the reverse complementary sequence of the adapter spacer sequence, the reverse complementary sequence of a linker spacer sequence and an A base from the 3' end to the 5' end. Wherein, the reverse complementary sequence of the linker spacer sequence is reverse complementary to the linker spacer sequence of the first strand of the linker oligonucleotide according to claim 26. Optionally, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25. Optionally, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27. Optionally, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26. The linker oligonucleotide is a Y-shaped linker oligonucleotide, that is, the upstream sequencing primer sequence and the reverse complementary sequence of the downstream sequencing primer sequence are not reverse complementary, and the upstream sequencing solid-phase binding sequence and the reverse complementary sequence of the downstream sequencing solid-phase binding sequence are not reverse complementary. Optionally, the first strand further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the reverse complementary sequence of the downstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. Wherein, the adaptor spacer sequence and the reverse complementary sequence of the adaptor spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

37. An adaptor oligonucleotide comprising a second strand, wherein, The second strand comprises, from the 3' end to the 5' end, the reverse complementary sequence of the downstream sequencing primer sequence, the reverse complementary sequence of the adaptor spacer sequence, and an A base. Wherein, the reverse complementary sequence of the adaptor spacer sequence is reverse complementary to the adaptor spacer sequence of the first strand of the adaptor oligonucleotide according to claim 29. Optionally, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26. The reverse complementary sequence of the downstream sequencing primer sequence is not reverse complementary to the upstream sequencing primer sequence of the adaptor oligonucleotide according to claim 29. Optionally, the second strand further comprises the reverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the reverse complementary sequence of the downstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

38. An adaptor oligonucleotide comprising a second strand, wherein, The second strand comprises, from the 3' end to the 5' end, the reverse complementary sequence of the downstream sequencing solid-phase binding sequence, the reverse complementary sequence of the downstream sequencing primer sequence, the reverse complementary sequence of the adaptor spacer sequence, and an A base. Wherein, the reverse complementary sequence of the adaptor spacer sequence is reverse complementary to the adaptor spacer sequence of the first strand of the adaptor oligonucleotide according to claim 32. Optionally, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24. Optionally, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26. The reverse complementary sequence of the downstream sequencing primer sequence is not reverse complementary to the upstream sequencing primer sequence of the adapter oligonucleotide according to claim 32, and the reverse complementary sequence of the downstream sequencing solid-phase binding sequence is not reverse complementary to the upstream sequencing solid-phase binding sequence of the adapter oligonucleotide according to claim 32. Optionally, the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3' end or 5' end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

39. A method for constructing a sequencing library, comprising: (1) Use a transposase complex encapsulated with the adapter oligonucleotide according to claim 23 to fragment the target sequence; (2) Treat with a DNA polymerase and ligase that are intolerant to damaged bases. Optionally, add the adaptor oligonucleotide according to claim 33 before ligase treatment and perform denaturation and annealing; and (3) Optionally, perform PCR using an extension primer pair. Wherein, the upstream primer of the extension primer pair comprises an upstream sequencing solid-phase binding sequence and the upstream sequencing primer sequence from the 5' end to the 3' end, and the downstream primer of the extension primer pair comprises a downstream sequencing solid-phase binding sequence and the downstream sequencing primer sequence from the 5' end to the 3' end. Optionally, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25. Optionally, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24. Optionally, the upstream sequencing solid-phase binding sequence is not reverse complementary to the reverse complementary sequence of the downstream sequencing solid-phase binding sequence when present. Optionally, the DNA polymerase that is intolerant to damaged bases is one or more of the DNA polymerases that are intolerant to U bases, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase, and Tgo DNA polymerase. Optionally, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, and Taq DNA ligase. Optionally, the upstream primer further comprises an upstream tag sequence located at the 5'-end or 3'-end of the upstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the downstream primer further comprises a downstream tag sequence located at the 5'-end or 3'-end of the downstream sequencing primer sequence, with a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

40. A method for constructing a sequencing library, comprising: (1) Fragment the target sequence using a transposase complex embedded with the adapter oligonucleotide according to claim 25; (2) Treat with a DNA polymerase intolerant to damaged bases, FEN1 enzyme and ligase, wherein the adapter oligonucleotide according to claim 35 is added and denatured and annealed before ligase treatment; and (3) Perform PCR using an extension primer pair. Wherein, the upstream primer of the extension primer pair comprises an upstream sequencing solid-phase binding sequence and the upstream sequencing primer sequence from the 5'-end to the 3'-end, and the downstream primer of the extension primer pair comprises a downstream sequencing solid-phase binding sequence and the downstream sequencing primer sequence from the 5'-end to the 3'-end. Optionally, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25. Optionally, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24. Optionally, when present, the upstream sequencing solid-phase binding sequence and the reverse complementary sequence of the downstream sequencing solid-phase binding sequence are not reverse complementary. Optionally, the DNA polymerase intolerant to damaged bases is one or more of the DNA polymerases intolerant to U bases, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase and Tgo DNA polymerase. Optionally, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase and Taq DNA ligase. Optionally, the upstream primer further comprises an upstream tag sequence, which is located at the 5' end or 3' end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the downstream primer further comprises a downstream tag sequence, which is located at the 5' end or 3' end of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

41. A method for constructing a sequencing library, comprising: (1) Using a transposase complex encapsulated with the adapter oligonucleotide according to claim 25 to fragment the target sequence; and (2) Treating with a DNA polymerase intolerant to damaged bases, FEN1 enzyme and ligase, wherein the adapter oligonucleotide according to claim 36 is added and denatured and annealed before the ligase treatment. Optionally, the DNA polymerase intolerant to damaged bases is one or more of DNA polymerases intolerant to U bases, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase and Tgo DNA polymerase. Optionally, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase and Taq DNA ligase.

42. A method for constructing a sequencing library, comprising: (1) Using a transposase complex encapsulated with the adapter oligonucleotide according to claim 26 to fragment the target sequence; (2) Treating with a DNA polymerase intolerant to damaged bases and a ligase, wherein the adapter oligonucleotide according to claim 35 is added before the ligase treatment; and (3) Performing PCR using an extension primer pair. Wherein, the upstream primer of the extension primer pair comprises an upstream sequencing solid-phase binding sequence and the upstream sequencing primer sequence from the 5' end to the 3' end, and the downstream primer of the extension primer pair comprises a downstream sequencing solid-phase binding sequence and the downstream sequencing primer sequence from the 5' end to the 3' end. Optionally, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25. Optionally, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24. Optionally, the upstream sequencing solid-phase binding sequence and the reverse complementary sequence of the downstream sequencing solid-phase binding sequence are not reverse complementary when present. Optionally, the DNA polymerase intolerant to damaged bases is one or more of the DNA polymerases intolerant to U bases, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase and Tgo DNA polymerase. Optionally, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase and Taq DNA ligase. Optionally, the upstream primer further comprises an upstream tag sequence, which is located at the 5' end or 3' end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the downstream primer further comprises a downstream tag sequence, which is located at the 5' end or 3' end of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

43. A method for constructing a sequencing library, comprising: (1) Disrupt the target sequence using a transposase complex encapsulated with the adaptor oligonucleotide according to claim 26; and (2) Treat with a DNA polymerase intolerant to damaged bases and a ligase, wherein the adaptor oligonucleotide according to claim 36 is added before the ligase treatment. Optionally, the DNA polymerase intolerant to damaged bases is one or more of the DNA polymerases intolerant to U bases, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase and Tgo DNA polymerase. Optionally, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase and Taq DNA ligase.

44. A method for constructing a sequencing library, comprising: (1) Disrupt the target sequence using a transposase complex encapsulated with the adaptor oligonucleotide according to claim 28; (2) Treat with a DNA polymerase intolerant to damaged bases, FEN1 enzyme and a ligase; and (3) Perform PCR using an extension primer pair. Wherein, the upstream primer of the extension primer pair comprises an upstream sequencing solid-phase binding sequence and the upstream sequencing primer sequence from the 5' end to the 3' end, and the downstream primer of the extension primer pair comprises a downstream sequencing solid-phase binding sequence and the downstream sequencing primer sequence from the 5' end to the 3' end. Optionally, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25. Optionally, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO. 25 or 24. Optionally, when the upstream sequencing solid-phase binding sequence and the reverse complementary sequence of the downstream sequencing solid-phase binding sequence are present, they are not reverse complementary. Optionally, the upstream primer further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. And / or, the downstream primer further comprises a downstream tag sequence, which is located at the 5'-end or 3'-end of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

45. A method for constructing a sequencing library, comprising: (1) Use a transposase complex embedded with the adaptor oligonucleotide according to claim 29 to fragment the target sequence. (2) Treat with a DNA polymerase and a ligase that are intolerant to damaged bases, wherein the adaptor oligonucleotide according to claim 37 is added before the ligase treatment; and (3) Perform PCR using an extension primer pair. Wherein, the upstream primer of the extension primer pair comprises an upstream sequencing solid-phase binding sequence and the upstream sequencing primer sequence from the 5'-end to the 3'-end, and the downstream primer of the extension primer pair comprises a downstream sequencing solid-phase binding sequence and the downstream sequencing primer sequence from the 5'-end to the 3'-end. Optionally, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO. 25 or 24. Optionally, when the upstream sequencing solid-phase binding sequence and the reverse complementary sequence of the downstream sequencing solid-phase binding sequence are present, they are not reverse complementary. Optionally, the DNA polymerase that is intolerant to damaged bases is one or more of the DNA polymerases that are intolerant to U bases, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, and Tgo DNA polymerase. Optionally, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, and Taq DNA ligase. Optionally, the upstream primer further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or, the downstream primer further comprises a downstream tag sequence, which is located at the 5'-end or 3'-end of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

46. A method for constructing a sequencing library, comprising: (1) Disrupting the target sequence using a transposase complex encapsulated with the adaptor oligonucleotide according to claim 31; and (2) Treating with a DNA polymerase intolerant to damaged bases, FEN1 enzyme and ligase, Optionally, the DNA polymerase intolerant to damaged bases is one or more of the DNA polymerases intolerant to U base, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase and Tgo DNA polymerase, Optionally, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase and Taq DNA ligase.

47. A method for constructing a sequencing library, comprising: (1) Disrupting the target sequence using a transposase complex encapsulated with the adaptor oligonucleotide according to claim 32; and (2) Treating with a DNA polymerase intolerant to damaged bases and a ligase, wherein the adaptor oligonucleotide according to claim 38 is added before the ligase treatment, Optionally, the DNA polymerase intolerant to damaged bases is one or more of the DNA polymerases intolerant to U base, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase and Tgo DNA polymerase, Optionally, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase and Taq DNA ligase.

48. A kit, which comprises (1) The adaptor oligonucleotide according to claim 25, the adaptor oligonucleotide according to claim 35 and an extension primer pair; (2) The adaptor oligonucleotide according to claim 25 and the adaptor oligonucleotide according to claim 36; (3) The adaptor oligonucleotide according to claim 26, the adaptor oligonucleotide according to claim 35 and an extension primer pair; (4) The adaptor oligonucleotide according to claim 26 and the adaptor oligonucleotide according to claim 36; (5) The adaptor oligonucleotide according to claim 28 and an extension primer pair; (6) The adaptor oligonucleotide according to claim 29, the adaptor oligonucleotide according to claim 37 and an extension primer pair; (7) The adaptor oligonucleotide according to claim 31; or The adapter oligonucleotide according to claim 32 and the linker oligonucleotide according to claim 38, Among them, The upstream primer of the extension primer pair comprises an upstream sequencing solid-phase binding sequence and the upstream sequencing primer sequence from the 5'-end to the 3'-end, and the downstream primer of the extension primer pair comprises a downstream sequencing solid-phase binding sequence and the downstream sequencing primer sequence from the 5'-end to the 3'-end. Optionally, the upstream primer further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the downstream primer further comprises a downstream tag sequence, which is located at the 5'-end or 3'-end of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. The kit further comprises one or more of the following: (1) transposase; (2) a DNA polymerase intolerant to damaged bases; and (3) ligase. Optionally, the DNA polymerase intolerant to damaged bases is one or more of the DNA polymerases intolerant to U base, including one or more of Klenow fragment, Pfu DNA polymerase, Vent DNA polymerase, Vent DNA polymerase and Tgo DNA polymerase. Optionally, the ligase is a DNA ligase, preferably one or more of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase and Taq DNA ligase.

49. An adapter oligonucleotide comprising a first strand and a second strand, wherein, The first strand comprises, from the 5'-end to the 3'-end, an optional upstream sequencing solid-phase binding sequence, an optional upstream sequencing primer sequence, an optional adapter spacer sequence, one or more restriction sequences I and a transposase recognition core sequence, and the second strand comprises, from the 3'-end to the 5'-end, the reverse complementary sequence of an optional downstream sequencing solid-phase binding sequence, the reverse complementary sequence of an optional downstream sequencing primer sequence, the reverse complementary sequence of an optional adapter spacer sequence, one or more restriction sequences II and the reverse complementary sequence of the transposase recognition core sequence, wherein the restriction sequence I and the restriction sequence II constitute the recognition / cutting site of a restriction endonuclease. The transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase, further optionally, it is the ME sequence, and further optionally, it is as shown in SEQ ID NO.

23. Optionally, the upstream sequencing solid-phase binding sequence is the sequencing solid-phase binding sequence of the Illumina platform, further optionally, it is P5 or P7 of the Illumina platform, and further optionally, it is as shown in SEQ ID NO.24 or 25. Optionally, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27. Optionally, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26. Optionally, the adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the reverse complementary sequence of the downstream sequencing primer sequence do not reverse complement when present, and the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the reverse complementary sequence of the downstream sequencing solid-phase binding sequence do not reverse complement when present. Optionally, the first strand further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3'-end or 5'-end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. Wherein, the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

50. An adaptor oligonucleotide comprising a first strand and a second strand, wherein, The first strand from the 5'-end to the 3'-end comprises an optional upstream sequencing solid-phase binding sequence, an upstream sequencing primer sequence, an optional linker spacer sequence, an optional adapter spacer sequence and restriction sequence three. The second strand from the 3'-end to the 5'-end comprises the reverse complementary sequence of an optional downstream sequencing solid-phase binding sequence, the reverse complementary sequence of a downstream sequencing primer sequence, the reverse complementary sequence of an optional linker spacer sequence, the reverse complementary sequence of an optional adapter spacer sequence and restriction sequence four. Wherein, the adapter spacer sequence is the same as the adapter spacer sequence recited in claim 49. Wherein, restriction sequence three and restriction sequence four constitute the sticky ends after cleavage by the restriction endonuclease recited in claim 49. Optionally, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25. Optionally, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27. Optionally, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26. The adapter oligonucleotide is a Y-shaped adapter oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence do not reverse complement when present, and the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence do not reverse complement when present. Optionally, the first strand further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3'-end or 5'-end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. Wherein, the adapter spacer sequence and the reverse complementary sequence of the adapter spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

51. An adaptor oligonucleotide comprising a first strand and a second strand, wherein, The first strand contains, from the 5'-end to the 3'-end, an upstream sequencing primer sequence, an optional adapter spacer sequence, an optional linker spacer sequence, and restriction sequence three. The second strand contains, from the 3'-end to the 5'-end, the reverse complementary sequence of the downstream sequencing primer sequence, the reverse complementary sequence of the optional adapter spacer sequence, the reverse complementary sequence of the optional linker spacer sequence, and restriction sequence four. Wherein, the linker spacer sequence is the same as the linker spacer sequence recited in claim 49. Wherein, restriction sequence three and restriction sequence four constitute the cohesive ends after cleavage by the restriction endonuclease recited in claim 49. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27. Optionally, the downstream sequencing primer sequence is a sequencing primer sequence for the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26. The adaptor oligonucleotide is a Y-shaped adaptor oligonucleotide, that is, the upstream sequencing primer sequence and the reverse complementary sequence of the downstream sequencing primer sequence are not reverse complementary. Optionally, the first strand further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3'-end or 5'-end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. Wherein, the adaptor spacer sequence and the reverse complementary sequence of the adaptor spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

52. An adaptor oligonucleotide comprising a first strand and a second strand, wherein, The first strand, from the 5'-end to the 3'-end, comprises an upstream sequencing solid-phase binding sequence, an upstream sequencing primer sequence, an optional adaptor spacer sequence, an optional linker spacer sequence, and restriction sequence three. The second strand, from the 3'-end to the 5'-end, comprises the reverse complementary sequence of a downstream sequencing solid-phase binding sequence, the reverse complementary sequence of a downstream sequencing primer sequence, an optional reverse complementary sequence of the adaptor spacer sequence, an optional reverse complementary sequence of the linker spacer sequence, and restriction sequence four. Wherein, the linker spacer sequence is the same as the linker spacer sequence recited in claim 49. Wherein, restriction sequence three and restriction sequence four constitute the sticky ends after cleavage by the restriction endonuclease recited in claim 49. Optionally, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence for the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25. Optionally, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence for the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence for the Illumina platform. Further optionally, it is read1 or read2 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.26 or 27. Optionally, the downstream sequencing primer sequence is a sequencing primer sequence for the Illumina platform. Further optionally, it is read2 or read1 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.27 or 26. The adaptor oligonucleotide is a Y-shaped adaptor oligonucleotide, that is, the reverse complementary sequence of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary, and the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence are not reverse complementary. Optionally, the first strand further comprises an upstream tag sequence, which is located at the 5' end or 3' end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the second strand further comprises the reverse complementary sequence of a downstream tag sequence, which is located at the 3' end or 5' end of the reverse complementary sequence of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. Wherein, the adaptor spacer sequence and the reverse complementary sequence of the adaptor spacer sequence have a length of at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

53. A method for constructing a sequencing library, comprising: (1) Use a transposase complex embedded with the adaptor oligonucleotide according to claim 49 to fragment the target sequence; (2) Use DNA polymerase, the restriction endonuclease according to claim 49 and ligase for treatment. Optionally, add the adaptor oligonucleotide according to claim 50 before ligase treatment and perform denaturation and annealing; and (3) Optionally, perform PCR using an extension primer pair. Wherein, the upstream primer of the extension primer pair comprises an upstream sequencing solid-phase binding sequence and the upstream sequencing primer sequence from the 5' end to the 3' end, and the downstream primer of the extension primer pair comprises a downstream sequencing solid-phase binding sequence and the downstream sequencing primer sequence from the 5' end to the 3' end. Optionally, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence for the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.24 or 25. Optionally, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence for the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO.25 or 24. Optionally, the reverse complementary sequence of the upstream sequencing solid-phase binding sequence and the downstream sequencing solid-phase binding sequence are not reverse complementary when present. Optionally, the upstream primer further comprises an upstream tag sequence, which is located at the 5' end or 3' end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the downstream primer further comprises a downstream tag sequence, which is located at the 5' end or 3' end of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

54. A method for constructing a sequencing library, comprising: (1) Using a transposase complex embedded with the adapter oligonucleotide according to claim 49 to break the target sequence; (2) Treating with a DNA polymerase, the restriction endonuclease according to claim 49 and a ligase, wherein the adapter oligonucleotide according to claim 51 is added before the ligase treatment; and (3) Performing PCR using an extension primer pair, wherein the upstream primer of the extension primer pair comprises an upstream sequencing solid-phase binding sequence and the upstream sequencing primer sequence from the 5' end to the 3' end, and the downstream primer of the extension primer pair comprises a downstream sequencing solid-phase binding sequence and the downstream sequencing primer sequence from the 5' end to the 3' end, Optionally, the upstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P5 or P7 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing solid-phase binding sequence is a sequencing solid-phase binding sequence of the Illumina platform. Further optionally, it is P7 or P5 of the Illumina platform. Further optionally, it is as shown in SEQ ID NO. 25 or 24. Optionally, the upstream sequencing solid-phase binding sequence and the reverse complementary sequence of the downstream sequencing solid-phase binding sequence do not reverse complement when present. Optionally, the upstream primer further comprises an upstream tag sequence, which is located at the 5' end or 3' end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the downstream primer further comprises a downstream tag sequence, which is located at the 5' end or 3' end of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides.

55. A method for constructing a sequencing library, comprising: (1) Using a transposase complex embedded with the adapter oligonucleotide according to claim 49 to break the target sequence; And (2) Treating with a DNA polymerase, the restriction endonuclease according to claim 49 and a ligase, wherein the adapter oligonucleotide according to claim 52 is added before the ligase treatment.

56. A kit, which comprises (1) The adapter oligonucleotide according to claim 49, the linker oligonucleotide according to claim 51, and the extension primer pair; or (2) The adapter oligonucleotide according to claim 49 and the linker oligonucleotide according to claim 52, Among them, The upstream primer of the extension primer pair comprises an upstream sequencing solid-phase binding sequence and the upstream sequencing primer sequence from the 5'-end to the 3'-end, and the downstream primer of the extension primer pair comprises a downstream sequencing solid-phase binding sequence and the downstream sequencing primer sequence from the 5'-end to the 3'-end. Optionally, the upstream primer further comprises an upstream tag sequence, which is located at the 5'-end or 3'-end of the upstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides, and / or the downstream primer further comprises a downstream tag sequence, which is located at the 5'-end or 3'-end of the downstream sequencing primer sequence and has a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. The kit further comprises one or more of the following: (1) transposase; (2) DNA polymerase; (3) the restriction endonuclease according to claim 49; and (4) ligase.

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