Adapter oligonucleotide, linker oligonucleotide, and sequencing library construction method

Abstract

Provided are an adapter oligonucleotide, a linker oligonucleotide, and a sequencing library construction method. By optimizing a transposase complex-embedded adapter sequence and / or linker sequence during transposase-based library construction, the loss caused by conventional transposase-based library construction can be reduced.

Description

Methods for constructing linker oligonucleotides, adaptor oligonucleotides, and sequencing libraries Technical Field

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

[0002] Next-generation sequencing (NGS) technology has been widely used in genome sequencing, disease diagnosis, transcriptome research, and other fields. Constructing a NGS library requires breaking DNA into appropriate lengths and then adding adapter sequences that can be recognized by the sequencing matrix and primers to both ends of the DNA fragments. Compared to traditional library construction methods, the Tn5 transposase method combines DNA fragmentation, trimming, and adapter ligation into a single step, resulting in a shorter experimental procedure, greater convenience, and significant time savings. Because Tn5 incorporates the two adapter sequences, adapter1 and adapter2, into the DNA fragment ends with equal probability, four possible outcomes exist at the ends of the DNA fragment: adapter1+adapter1, adapter1+adapter2, adapter2+adapter1, and adapter2+adapter2. The DNA molecule must contain different adapter sequences at both ends for correct amplification and sequencing; therefore, theoretically, 50% of the yield will be lost.

[0003] Chinese patent CN109154013B discloses a complex composed of a transposase and a Y-adaptor for fragmenting and tagging DNA. After Y-adaptor sequences are inserted at both ends of the DNA, the gaps are filled using a polymerase under non-strand displacement conditions, and then ligase completes the closure. Alternatively, a strand displacement polymerase can replace some nucleotides in the downstream double-stranded region; the resulting structure can be eliminated using a valve-shaped endonuclease, and then ligase completes the gap closure, thereby producing a library with different adapter sequences at both ends to compensate for 50% loss.

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

[0005] However, the inventors of this application discovered during the actual application of complexes composed of Y-adaptors that nucleotide chain substitution cannot be completely avoided when using non-chain substitution conditions. Even when using chain substitution conditions, it is not just a few nucleotides that undergo chain substitution. The actual chain substitution process has a certain degree of uncontrollability. In some cases, the P7 sequence may be completely substituted, and the polymerase may extend to produce a complementary sequence to the P5 sequence. As a result, P5 sequences are inserted at both ends of the DNA, leading to incorrect amplification. This method can only partially compensate for 50% of the lost DNA, and there is still room for improvement in library yield.

[0006] In addition, while the adapter replacement strategy theoretically avoids library loss caused by the uncontrollable chain replacement process, the overall library construction process involves multiple openings to add reagents and mix, which is complex, time-consuming, and results in significant library loss. Summary of the Invention

[0007] The purpose of this application is to provide a linker oligonucleotide, an adaptor oligonucleotide, and a sequencing library construction method. This method optimizes the linker sequence embedded in the transposase and designs corresponding adaptors, enabling different adaptor sequences to be attached to both ends of the DNA. This solves the problem of 50% DNA fragment loss during traditional Tn5 transposase library construction, thus improving library yield.

[0008] A first aspect of this 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 optional upstream sequencing immobilization sequence, an optional upstream sequencing primer sequence, and a transposase recognition core sequence, and the second strand comprises, from the 3' end to the 5' end, an inverse complementary sequence of an optional downstream sequencing immobilization sequence, an inverse complementary sequence of an optional downstream sequencing primer sequence, and an inverse complementary sequence of the transposase recognition core sequence.

[0009] In this embodiment, 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 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.

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

[0012] In some embodiments, the methylated base is, for example, any one of N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymidine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymidine; and the oxidized base is, for example, any one of 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 the Tn5 transposase, and more optionally, it is the ME sequence, and more optionally, it is as shown in SEQ ID NO.23.

[0014] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0015] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more optionally, it is as shown in SEQ ID NO. 25 or 24.

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

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

[0018] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0019] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0020] In some implementations, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0021] In some implementations, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0022] In some implementations, the linker oligonucleotide is a Y-type linker oligonucleotide, that is, the reverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary when they are present, and the reverse complementary sequences of the upstream sequencing binding sequence and the downstream sequencing binding sequence are not reverse complementary when they are present.

[0023] In some embodiments, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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.

[0024] In some implementations, the linker oligonucleotide may have a linker spacer sequence added to the 5' end of the ME sequence, especially when U is close to the 5' end; correspondingly, the adaptor oligonucleotide needs to have a corresponding sequence added, and attention should be paid to the competition between the linker spacer sequence and the adaptor spacer sequence.

[0025] In some embodiments, the first strand further comprises a linker spacer sequence located at the 5' end of the transposase recognition core sequence. Optionally, the second strand further comprises an inverse complementary sequence of the linker spacer sequence located at the 3' end of the inverse complementary sequence of the transposase recognition core sequence. The linker spacer sequence and its inverse complementary sequence are at least 1, 2, 3, 4, 5, 6, or more nucleotides in length.

[0026] A second aspect of this application provides a linker oligonucleotide comprising a first strand and a second strand, wherein the first strand contains a transposase-recognizing core sequence, and the second strand contains the inverse complementary sequence of the transposase-recognizing core sequence.

[0027] In this embodiment, 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 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.

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

[0030] In some embodiments, the methylated base is, for example, any one of N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymidine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymidine; and the oxidized base is, for example, any one of 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 the Tn5 transposase, and more optionally, it is the ME sequence, and more optionally, it is as shown in SEQ ID NO.23.

[0032] A third aspect of this 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 primer sequence and a transposase recognition core sequence, and the second strand comprises, from the 3' end to the 5' end, an inverse complementary sequence of an optional downstream sequencing primer sequence and an inverse complementary sequence of the transposase recognition core sequence.

[0033] In this embodiment, 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 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.

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

[0036] In some embodiments, the methylated base is, for example, any one of N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymidine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymidine; and the oxidized base is, for example, any one of 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 the Tn5 transposase, and more optionally, it is the ME sequence, and more optionally, it is as shown in SEQ ID NO.23.

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

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

[0040] In some implementations, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0041] In some implementations, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0042] In some implementations, the linker oligonucleotide is a Y-type linker oligonucleotide, meaning that the reverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary when present.

[0043] In some embodiments, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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.

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

[0045] In this embodiment, 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 a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and baseless site, preferably a U base.

[0048] In some embodiments, the methylated base is, for example, any one of N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymidine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymidine; and the oxidized base is, for example, any one of 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 the Tn5 transposase, and more optionally, it is the ME sequence, and more optionally, it is as shown in SEQ ID NO.23.

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

[0052] In some implementations, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0053] In some implementations, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0054] In some implementations, the linker oligonucleotide is a Y-type linker oligonucleotide, meaning that the reverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary.

[0055] In some embodiments, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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.

[0056] A fifth aspect of this application provides 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 the transposase recognition core sequence.

[0057] In this embodiment, 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 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.

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

[0060] In some embodiments, the methylated base is, for example, any one of N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymidine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymidine; and the oxidized base is, for example, any one of 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 the Tn5 transposase, and more optionally, it is the ME sequence, and more optionally, it is as shown in SEQ ID NO.23.

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

[0063] In some implementations, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0064] In some implementations, the first chain further includes an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, the upstream tag sequence 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.

[0065] A sixth aspect of this 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 immobilization sequence, an upstream sequencing primer sequence, and a transposase recognition core sequence, and the second strand comprises, from the 3' end to the 5' end, an inverse complementary sequence of an optional downstream sequencing immobilization sequence, an inverse complementary sequence of an optional downstream sequencing primer sequence, and an inverse complementary sequence of the transposase recognition core sequence.

[0066] In this embodiment, 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 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.

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

[0069] In some embodiments, the methylated base is, for example, any one of N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymidine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymidine; and the oxidized base is, for example, any one of 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 the Tn5 transposase, and more optionally, it is the ME sequence, and more optionally, it is as shown in SEQ ID NO.23.

[0071] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0072] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more optionally, it is as shown in SEQ ID NO. 25 or 24.

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

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

[0075] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0076] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0077] In some implementations, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0078] In some implementations, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0079] In some implementations, the linker oligonucleotide is a Y-type linker oligonucleotide, that is, the reverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary when they are present, and the reverse complementary sequences of the upstream sequencing binding sequence and the downstream sequencing binding sequence are not reverse complementary when they are present.

[0080] In some embodiments, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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.

[0081] A seventh aspect of this 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 immobilization sequence, an upstream sequencing primer sequence, and a transposase recognition core sequence, and the second strand comprises, from the 3' end to the 5' end, an inverse complementary sequence of the downstream sequencing immobilization sequence, an inverse complementary sequence of the downstream sequencing primer sequence, and an inverse complementary sequence of the transposase recognition core sequence.

[0082] In this embodiment, 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 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.

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

[0085] In some embodiments, the methylated base is, for example, any one of N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymidine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymidine; and the oxidized base is, for example, any one of 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 the Tn5 transposase, and more optionally, it is the ME sequence, and more optionally, it is as shown in SEQ ID NO.23.

[0087] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0088] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more 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, and more optionally, it is read1 or read2 of the Illumina platform, and more 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, and more optionally, it is read2 or read1 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 27 or 26.

[0091] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0092] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0093] In some implementations, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0094] In some implementations, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0095] In some implementations, the linker oligonucleotide is a Y-type linker oligonucleotide, that is, the reverse complementary sequences 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 binding sequence and the downstream sequencing binding sequence are not reverse complementary.

[0096] In some embodiments, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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.

[0097] An eighth aspect of this 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 immobilization sequence, an upstream sequencing primer sequence, and a transposase recognition core sequence, and the second strand comprises the reverse complementary sequence of the transposase recognition core sequence.

[0098] In this embodiment, 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 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.

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

[0101] In some embodiments, the methylated base is, for example, any one of N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymidine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymidine; and the oxidized base is, for example, any one of 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 the Tn5 transposase, and more optionally, it is the ME sequence, and more optionally, it is as shown in SEQ ID NO.23.

[0103] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more 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, and more optionally, it is read1 or read2 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 26 or 27.

[0105] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0106] In some implementations, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0107] In some implementations, the first chain further includes an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, the upstream tag sequence 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.

[0108] A ninth aspect of this application provides an adaptor oligonucleotide comprising 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 immobilization sequence, an upstream sequencing primer sequence, and an adaptor spacer sequence; and the second strand comprises, from the 3' end to the 5' end, an optional reverse complementary sequence of a downstream sequencing immobilization sequence, an reverse complementary sequence of a downstream sequencing primer sequence, an optional reverse complementary sequence of the adaptor spacer sequence, the 3' portion of the reverse complementary sequence of a transposase recognition core sequence, and an A base.

[0109] Wherein, the 3' end portion of the inverse complementary sequence of the transposase recognition core sequence is inversely 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 implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0111] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more 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, and more optionally, it is read1 or read2 of the Illumina platform, and more 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, and more optionally, it is read2 or read1 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 27 or 26.

[0114] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0115] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0116] In some implementations, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0117] In some implementations, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0118] In some implementations, the adaptor oligonucleotide is a Y-type adaptor oligonucleotide, that is, the reverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary when they are present, and the reverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not reverse complementary when they are present.

[0119] In some embodiments, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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, wherein the length of the adaptor spacer sequence and the inverse complementary sequence of the adaptor spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0120] A tenth aspect of this application provides an adaptor 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 immobilization sequence, an upstream sequencing primer sequence, and an adaptor spacer sequence; and the second strand comprises, from the 3' end to the 5' end, an optional reverse complementary sequence of a downstream sequencing immobilization sequence, an reverse complementary sequence of a downstream sequencing primer sequence, an reverse complementary sequence of the adaptor spacer sequence, the 3' portion of the reverse complementary sequence of a transposase recognition core sequence, and an A base.

[0121] Wherein, the 3' end portion of the inverse complementary sequence of the transposase recognition core sequence is inversely 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, 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.

[0122] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0123] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more 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, and more optionally, it is read1 or read2 of the Illumina platform, and more 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 of the Illumina platform, and more optionally, it is read2 or read1 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 27 or 26.

[0126] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0127] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0128] In some implementations, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0129] In some implementations, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0130] In some implementations, the adaptor oligonucleotide is a Y-type adaptor oligonucleotide, that is, the reverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary when they are present, and the reverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not reverse complementary when they are present.

[0131] In some embodiments, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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, wherein the length of the adaptor spacer sequence and the inverse complementary sequence of the adaptor spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0132] The eleventh invention of this application provides an adaptor 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 primer sequence and an adaptor spacer sequence, and the second strand comprises, from the 3' end to the 5' end, an inverse complementary sequence of a downstream sequencing primer sequence, an inverse complementary sequence of the adaptor spacer sequence, a 3' portion of an inverse complementary sequence of a transposase recognition core sequence, and an A base.

[0133] Wherein, the 3' end portion of the inverse complementary sequence of the transposase recognition core sequence is inversely 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, 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.

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

[0136] In some implementations, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0137] In some implementations, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0138] In some embodiments, the adaptor oligonucleotide is a Y-type adaptor oligonucleotide, that is, the reverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary. Optionally, the first strand further includes 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 includes a reverse complementary sequence of the 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 length of the reverse complementary sequence of the adaptor spacer sequence and the adaptor spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0139] The twelfth invention of this application provides an adaptor 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 immobilization sequence, an upstream sequencing primer sequence, and an adaptor spacer sequence; and the second strand comprises, from the 3' end to the 5' end, an inverse complementary sequence of the downstream sequencing immobilization sequence, an inverse complementary sequence of the downstream sequencing primer sequence, an inverse complementary sequence of the adaptor spacer sequence, the 3' end portion of the inverse complementary sequence of the transposase recognition core sequence, and an A base.

[0140] Wherein, the 3' end portion of the inverse complementary sequence of the transposase recognition core sequence is inversely 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, 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.

[0141] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0142] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more optionally, it is as shown in SEQ ID NO. 25 or 24.

[0143] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and more optionally, it is read1 or read2 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 26 or 27.

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

[0145] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0146] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0147] In some implementations, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0148] In some implementations, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0149] In some implementations, the adaptor oligonucleotide is a Y-type adaptor oligonucleotide, that is, the reverse complementary sequences 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 binding sequence and the downstream sequencing binding sequence are not reverse complementary.

[0150] In some embodiments, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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, wherein the length of the adaptor spacer sequence and the inverse complementary sequence of the adaptor spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0151] The thirteenth aspect of this application provides an adaptor oligonucleotide comprising a second strand, wherein the second strand comprises, from the 3' end to the 5' end, an inverse complementary sequence of a downstream sequencing primer sequence, a 3' portion of an inverse complementary sequence of a transposase recognition core sequence, and an A base.

[0152] Wherein, the 3' end portion of the inverse complementary sequence of the transposase recognition core sequence is inversely 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.

[0153] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and more optionally, it is read2 or read1 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 27 or 26.

[0154] In some implementations, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0155] In some implementations, the reverse complementary sequence of the downstream sequencing primer sequence is not reverse complementary to the upstream sequencing primer sequence of the linker oligonucleotide described in the fifth aspect.

[0156] In some embodiments, the second strand further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, the downstream tag sequence 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.

[0157] The fourteenth aspect of this application provides an adaptor oligonucleotide comprising a second strand, wherein the second strand comprises, from the 3' end to the 5' end, an inverse complementary sequence to a downstream sequencing primer sequence, an inverse complementary sequence to a transposase-recognized core sequence, a 3' portion thereof, and an A base.

[0158] Wherein, the 3' end portion of the inverse complementary sequence of the transposase recognition core sequence is inversely complementary to the 5' end portion of the transposase recognition core sequence of the first strand of the linker oligonucleotide according to the eighth 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.

[0159] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more optionally, it is as shown in SEQ ID NO. 25 or 24.

[0160] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and more optionally, it is read2 or read1 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 27 or 26.

[0161] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0162] In some implementations, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0163] 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 eighth aspect, and the reverse complementary sequence of the downstream sequencing immobilization sequence is not reverse complementary to the upstream sequencing immobilization sequence of the linker oligonucleotide according to the eighth aspect.

[0164] In some embodiments, the second strand further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, the downstream tag sequence 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.

[0165] The fifteenth aspect of this application provides a method for constructing a sequencing library, comprising: (1) breaking a target sequence using a transposase complex embedded with an adapter oligonucleotide as described in the first aspect; (2) treating the target sequence with a DNA polymerase, FEN1 enzyme, and ligase that are intolerant to base damage, optionally adding an adapter oligonucleotide as described in the ninth aspect and denaturing and annealing the target sequence before ligase treatment; and (3) optionally performing PCR using an extension primer pair.

[0166] The upstream primer of the extension primer pair contains an upstream sequencing immobilization 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 immobilization sequence and the downstream sequencing primer sequence from the 5' end to the 3' end.

[0167] 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.

[0168] In some embodiments, the DNA polymerase that is intolerant to damaged bases is a hypoxanthine-intolerant Pfu DNA polymerase or a Deep vent DNA polymerase.

[0169] 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.

[0170] In some embodiments, the FEN1 enzyme includes, but is not limited to, wild-type, mutant, homologous, and isoenzyme of the FEN1 enzyme, such as Rad27 / FEN1 and Rad2 / FEN1.

[0171] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0172] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more optionally, it is as shown in SEQ ID NO. 25 or 24.

[0173] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0174] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0175] In some implementations, the upstream sequencing binding sequence and the inverse complementary sequence of the downstream sequencing binding sequence are not inversely complementary when they are present.

[0176] In some embodiments, the upstream primer further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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' or 3' end of the downstream sequencing primer sequence, 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.

[0177] The sixteenth aspect of this application provides a method for constructing a sequencing library, comprising: (1) breaking a target sequence using a transposase complex embedded with an adapter oligonucleotide as described in the second aspect; (2) treating the target sequence with a DNA polymerase, FEN1 enzyme, and ligase that are intolerant to base damage, wherein the adapter oligonucleotide as described in the eleventh aspect is added and denatured and annealed before ligase treatment; and (3) performing PCR using an extension primer pair.

[0178] The upstream primer of the extension primer pair contains an upstream sequencing immobilization 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 immobilization sequence and the downstream sequencing primer sequence from the 5' end to the 3' end.

[0179] 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.

[0180] In some embodiments, the DNA polymerase that is intolerant to damaged bases is a hypoxanthine-intolerant Pfu DNA polymerase or a Deep vent DNA polymerase.

[0181] 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.

[0182] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0183] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more optionally, it is as shown in SEQ ID NO. 25 or 24.

[0184] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0185] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0186] In some implementations, the upstream sequencing binding sequence and the inverse complementary sequence of the downstream sequencing binding sequence are not inversely complementary when present.

[0187] In some embodiments, the upstream primer further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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' or 3' end of the downstream sequencing primer sequence, 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.

[0188] The seventeenth aspect of this application provides a method for constructing a sequencing library, comprising: (1) breaking a target sequence using a transposase complex containing an adapter oligonucleotide as described in the second aspect; and (2) treating the sequence with a DNA polymerase, FEN1 enzyme, and ligase that are intolerant to base damage, wherein the adapter oligonucleotide as described in the twelfth aspect is added and denatured and annealed prior to the ligase treatment.

[0189] 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.

[0190] In some embodiments, the DNA polymerase that is intolerant to damaged bases is a hypoxanthine-intolerant Pfu DNA polymerase or a Deep vent DNA polymerase.

[0191] In some embodiments, the FEN1 enzyme includes, but is not limited to, wild-type, mutant, homologous, and isoenzyme of the FEN1 enzyme, such as Rad27 / FEN1 and Rad2 / FEN1.

[0192] 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.

[0193] The eighteenth aspect of this application provides a method for constructing a sequencing library, comprising: (1) breaking a target sequence using a transposase complex embedded with a linker oligonucleotide as described in the fourth aspect; (2) treating the sequence with a DNA polymerase, FEN1 enzyme, and ligase that are intolerant to base damage; and (3) performing PCR using an extension primer pair.

[0194] The upstream primer of the extension primer pair contains an upstream sequencing immobilization 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 immobilization sequence and the downstream sequencing primer sequence from the 5' end to the 3' end.

[0195] 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.

[0196] In some embodiments, the DNA polymerase that is intolerant to damaged bases is a hypoxanthine-intolerant Pfu DNA polymerase or a Deep vent DNA polymerase.

[0197] 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.

[0198] In some implementations, a polymerase for the bases intolerant to damage is used to fill the 9-base gap between the linker oligonucleotide and the target sequence. Then, strand substitution is performed, and the strand substitution reaction is stopped when it reaches the damaged base site, resulting in a forked double-stranded DNA structure. The 5'Flap structure in the forked double-stranded DNA structure is removed using the FEN1 enzyme to create a gap. The gap is then closed using a ligase, so that different linker sequences are attached to both ends of the target sequence, as shown in Figure 9.

[0199] In some embodiments, the FEN1 enzyme includes, but is not limited to, wild-type, mutant, homologous, and isoenzyme of the FEN1 enzyme, such as Rad27 / FEN1 and Rad2 / FEN1.

[0200] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0201] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more optionally, it is as shown in SEQ ID NO. 25 or 24.

[0202] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0203] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0204] In some implementations, the upstream sequencing binding sequence and the inverse complementary sequence of the downstream sequencing binding sequence are not inversely complementary when they are present.

[0205] In some embodiments, the upstream primer further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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' or 3' end of the downstream sequencing primer sequence, 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.

[0206] The nineteenth aspect of this application provides a method for constructing a sequencing library, comprising: (1) breaking a target sequence using a transposase complex embedded with an adapter oligonucleotide as described in the fifth aspect; (2) treating the sequence with a DNA polymerase, FEN1 enzyme, and ligase that are intolerant to base damage, wherein an adapter oligonucleotide as described in the thirteenth aspect is added and denatured and annealed before ligase treatment; and (3) performing PCR using an extension primer pair.

[0207] The upstream primer of the extension primer pair contains an upstream sequencing immobilization 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 immobilization sequence and the downstream sequencing primer sequence from the 5' end to the 3' end.

[0208] 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.

[0209] In some embodiments, the DNA polymerase that is intolerant to damaged bases is a hypoxanthine-intolerant Pfu DNA polymerase or a Deep vent DNA polymerase.

[0210] 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.

[0211] In some embodiments, the FEN1 enzyme includes, but is not limited to, wild-type, mutant, homologous, and isoenzyme of the FEN1 enzyme, such as Rad27 / FEN1 and Rad2 / FEN1.

[0212] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0213] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more optionally, it is as shown in SEQ ID NO. 25 or 24.

[0214] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0215] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0216] In some implementations, the upstream sequencing binding sequence and the inverse complementary sequence of the downstream sequencing binding sequence are not inversely complementary when present.

[0217] In some embodiments, the upstream primer further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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' or 3' end of the downstream sequencing primer sequence, 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.

[0218] The twentieth aspect of this application provides a method for constructing a sequencing library, comprising: (1) breaking a target sequence using a transposase complex containing a linker oligonucleotide as described in the seventh aspect; and (2) treating the sequence with a DNA polymerase, FEN1 enzyme, and ligase that are intolerant to base damage.

[0219] 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.

[0220] In some embodiments, the DNA polymerase that is intolerant to damaged bases is a hypoxanthine-intolerant Pfu DNA polymerase or a Deep vent DNA polymerase.

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

[0222] In some embodiments, a polymerase for the bases intolerant to damage is used to fill the 9-base gap between the linker oligonucleotide and the target sequence, followed by strand substitution. The strand substitution reaction is stopped when it reaches the damaged base site, resulting in a forked double-stranded DNA structure. The 5'Flap structure in the forked double-stranded DNA structure is removed using the FEN1 enzyme, creating a gap. The gap is then closed using a ligase, resulting in different linker sequences at both ends of the target sequence.

[0223] In some embodiments, the FEN1 enzyme includes, but is not limited to, wild-type, mutant, homologous, and isoenzyme of the FEN1 enzyme, such as Rad27 / FEN1 and Rad2 / FEN1.

[0224] 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.

[0225] The 21st aspect of this application provides a method for constructing a sequencing library, comprising: (1) breaking a target sequence using a transposase complex containing an adapter oligonucleotide as described in the 8th aspect; and (2) treating the sequence with a loss-intolerant DNA polymerase, FEN1 enzyme, and ligase, wherein the adapter oligonucleotide as described in the 14th aspect is added and denatured and annealed prior to the ligase treatment.

[0226] 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.

[0227] In some embodiments, the DNA polymerase that is intolerant to damaged bases is a hypoxanthine-intolerant Pfu DNA polymerase or a Deep vent DNA polymerase.

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

[0229] In some embodiments, the FEN1 enzyme includes, but is not limited to, wild-type, mutant, homologous, and isoenzyme of the FEN1 enzyme, such as Rad27 / FEN1 and Rad2 / FEN1.

[0230] 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.

[0231] The 22nd aspect of this application provides a kit comprising...

[0232] (1) The linker oligonucleotide according to the second aspect, the integrator oligonucleotide according to the eleventh aspect, and the extension primer pair;

[0233] (2) The linker oligonucleotide according to the second aspect and the integrator oligonucleotide according to the twelfth aspect;

[0234] (3) The linker oligonucleotide and extension primer pair as described in the fourth aspect;

[0235] (4) The linker oligonucleotide as described in the fifth aspect, the integrator oligonucleotide as described in the thirteenth aspect, and the extension primer pair;

[0236] (5) The linker oligonucleotide as described in aspect seven; or

[0237] (6) The linker oligonucleotides according to aspect eight and the integrator oligonucleotides according to aspect fourteen.

[0238] The upstream primer of the extension primer pair contains an upstream sequencing immobilization 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 immobilization sequence and the downstream sequencing primer sequence from the 5' end to the 3' end.

[0239] In some embodiments, the upstream primer further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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' or 3' end of the downstream sequencing primer sequence, 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.

[0240] In some embodiments, the kit further comprises one or more of the following: (1) a transposase; (2) a DNA polymerase intolerant to base damage; (3) a FEN1 enzyme; and (4) a ligase.

[0241] 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.

[0242] In some embodiments, the DNA polymerase intolerant to damaged bases is a hypoxanthine-intolerant Pfu DNA polymerase or a Deep vent DNA polymerase.

[0243] In some embodiments, the FEN1 enzyme includes, but is not limited to, wild-type, mutant, homologous protein and isoenzyme of the FEN1 enzyme, such as Rad27 / FEN1 and Rad2 / FEN1.

[0244] 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.

[0245] The 23rd aspect of this 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 optional upstream sequencing immobilization sequence, an optional upstream sequencing primer sequence, a linker spacer sequence, one or more damaged bases, and a transposase recognition core sequence; and the second strand comprises, from the 3' end to the 5' end, an optional reverse complementary sequence of a downstream sequencing immobilization sequence, an optional reverse complementary sequence of a downstream sequencing primer sequence, an optional reverse complementary sequence of the linker spacer sequence, an optional one or more A bases, and an reverse complementary sequence of the transposase recognition core sequence.

[0246] The length of the linker spacer sequence and its inverse complementary sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0247] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase, and more optionally, it is the ME sequence, and more optionally, it is as shown in SEQ ID NO.23.

[0248] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0249] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more optionally, it is as shown in SEQ ID NO. 25 or 24.

[0250] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and more optionally, it is read1 or read2 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 26 or 27.

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

[0252] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0253] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0254] In some implementations, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0255] In some implementations, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0256] In some implementations, the linker oligonucleotide is a Y-type linker oligonucleotide, that is, the reverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary when they are present, and the reverse complementary sequences of the upstream sequencing binding sequence and the downstream sequencing binding sequence are not reverse complementary when they are present.

[0257] In some embodiments, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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.

[0258] In some embodiments, the damaged base is any one of a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and baseless site, preferably a U base.

[0259] In some embodiments, the methylated base is, for example, any one of N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymidine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymidine; and the oxidized base is, for example, any one of 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA, and Fapy-dG.

[0260] In some embodiments, the number of damaged bases is at most 20, preferably at most 10, for example 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.

[0261] In some implementations, the linker oligonucleotide may have a linker spacer sequence added to the 5' end of the ME sequence.

[0262] In some embodiments, the first strand further comprises a linker spacer sequence located at the 5' end of the transposase recognition core sequence. Optionally, the second strand further comprises an inverse complementary sequence of the linker spacer sequence located at the 3' end of the inverse complementary sequence of the transposase recognition core sequence. The linker spacer sequence and its inverse complementary sequence are at least 1, 2, 3, 4, 5, 6, or more nucleotides in length.

[0263] The 24th aspect of this 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, a linker spacer sequence, a damaged base, and a transposase recognition core sequence, and the second strand comprises, from the 3' end to the 5' end, an optional inverse complementary sequence of the linker spacer sequence, an optional A base, and an inverse complementary sequence of the transposase recognition core sequence.

[0264] The length of the linker spacer sequence and its inverse complementary sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0265] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase, and more optionally, it is the ME sequence, and more optionally, it is as shown in SEQ ID NO.23.

[0266] In some embodiments, the damaged base is any one of a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and baseless site, preferably a U base.

[0267] In some embodiments, the methylated base is, for example, any one of N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymidine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymidine; and the oxidized base is, for example, any one of 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA, and Fapy-dG.

[0268] The 25th aspect of this 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, a linker spacer sequence, a damaged base, and a transposase recognition core sequence, and the second strand comprises, from the 3' end to the 5' end, an inverse complementary sequence of the linker spacer sequence, an A base, and an inverse complementary sequence of the transposase recognition core sequence.

[0269] The length of the linker spacer sequence and its inverse complementary sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0270] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase, and more optionally, it is the ME sequence, and more optionally, it is as shown in SEQ ID NO.23.

[0271] In some embodiments, the damaged base is any one of a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and baseless site, preferably a U base.

[0272] In some embodiments, the methylated base is, for example, any one of N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymidine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymidine; and the oxidized base is, for example, any one of 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA, and Fapy-dG.

[0273] The 26th aspect of this 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, a linker spacer sequence, a damaged base, and a transposase recognition core sequence, and the second strand comprises the reverse complementary sequence of the transposase recognition core sequence.

[0274] The length of the linker spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0275] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase, and more optionally, it is the ME sequence, and more optionally, it is as shown in SEQ ID NO.23.

[0276] In some embodiments, the damaged base is any one of a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and baseless site, preferably a U base.

[0277] In some embodiments, the methylated base is, for example, any one of N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymidine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymidine; and the oxidized base is, for example, any one of 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA, and Fapy-dG.

[0278] The 27th aspect of this application discloses 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 primer sequence, a linker spacer sequence, a damaged base, and a transposase recognition core sequence, and the second strand comprises, from the 3' end to the 5' end, an optional reverse complementary sequence of a downstream sequencing primer sequence, an optional reverse complementary sequence of the linker spacer sequence, an optional A base, and an reverse complementary sequence of the transposase recognition core sequence.

[0279] The length of the linker spacer sequence and its inverse complementary sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0280] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase, and more optionally, it is the ME sequence, and more optionally, it is as shown in SEQ ID NO.23.

[0281] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and more optionally, it is read1 or read2 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 26 or 27.

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

[0283] In some implementations, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0284] In some implementations, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0285] In some implementations, the linker oligonucleotide is a Y-type linker oligonucleotide, meaning that the reverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary when present.

[0286] 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, the upstream tag sequence being at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides in length, and / or the second strand further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, the downstream tag sequence being at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides in length.

[0287] In some embodiments, the damaged base is any one of a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and baseless site, preferably a U base.

[0288] In some embodiments, the methylated base is, for example, any one of N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymidine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymidine; and the oxidized base is, for example, any one of 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA, and Fapy-dG.

[0289] The 28th aspect of this 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 primer sequence, a linker spacer sequence, a damaged base, and a transposase recognition core sequence, and the second strand comprises, from the 3' end to the 5' end, an inverse complementary sequence of the downstream sequencing primer sequence, an inverse complementary sequence of the linker spacer sequence, an A base, and an inverse complementary sequence of the transposase recognition core sequence.

[0290] The length of the linker spacer sequence and its inverse complementary sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0291] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase, and more optionally, it is the ME sequence, and more optionally, it is as shown in SEQ ID NO.23.

[0292] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and more optionally, it is read1 or read2 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 26 or 27.

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

[0294] In some implementations, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0295] In some implementations, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0296] In some implementations, the linker oligonucleotide is a Y-type linker oligonucleotide, meaning that the reverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary.

[0297] In some embodiments, the damaged base is any one of a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and baseless site, preferably a U base.

[0298] In some embodiments, the methylated base is, for example, any one of N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymidine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymidine; and the oxidized base is, for example, any one of 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA, and Fapy-dG.

[0299] In some embodiments, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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.

[0300] The 29th aspect of this 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 primer sequence, a linker spacer sequence, a damaged base, and a transposase recognition core sequence, and the second strand comprises the reverse complementary sequence of the transposase recognition core sequence.

[0301] The length of the linker spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0302] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase, and more optionally, it is the ME sequence, and more optionally, it is as shown in SEQ ID NO.23.

[0303] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and more optionally, it is read1 or read2 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 26 or 27.

[0304] In some implementations, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0305] In some embodiments, the damaged base is any one of a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and baseless site, preferably a U base.

[0306] In some embodiments, the methylated base is, for example, any one of N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymidine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymidine; and the oxidized base is, for example, any one of 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA, and Fapy-dG.

[0307] In some implementations, the first chain further includes an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, the upstream tag sequence 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.

[0308] A thirtieth aspect of this 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 immobilization sequence, an upstream sequencing primer sequence, a linker spacer sequence, a damaged base, and a transposase recognition core sequence; and the second strand comprises, from the 3' end to the 5' end, an optional reverse complementary sequence of a downstream sequencing immobilization sequence, an optional reverse complementary sequence of a downstream sequencing primer sequence, an optional reverse complementary sequence of the linker spacer sequence, an optional A base, and an optional reverse complementary sequence of the transposase recognition core sequence.

[0309] The length of the linker spacer sequence and its inverse complementary sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0310] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase, and more optionally, it is the ME sequence, and more optionally, it is as shown in SEQ ID NO.23.

[0311] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0312] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more optionally, it is as shown in SEQ ID NO. 25 or 24.

[0313] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and more optionally, it is read1 or read2 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 26 or 27.

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

[0315] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0316] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0317] In some implementations, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0318] In some implementations, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0319] In some implementations, the linker oligonucleotide is a Y-type linker oligonucleotide, that is, the reverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary when they are present, and the reverse complementary sequences of the upstream sequencing binding sequence and the downstream sequencing binding sequence are not reverse complementary when they are present.

[0320] In some embodiments, the damaged base is any one of a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and baseless site, preferably a U base.

[0321] In some embodiments, the methylated base is, for example, any one of N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymidine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymidine; and the oxidized base is, for example, any one of 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA, and Fapy-dG.

[0322] In some embodiments, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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.

[0323] The thirty-first aspect of this 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 immobilization sequence, an upstream sequencing primer sequence, a linker spacer sequence, a damaged base, and a transposase recognition core sequence; and the second strand comprises, from the 3' end to the 5' end, an inverse complementary sequence of the downstream sequencing immobilization sequence, an inverse complementary sequence of the downstream sequencing primer sequence, an inverse complementary sequence of the linker spacer sequence, an A base, and an inverse complementary sequence of the transposase recognition core sequence.

[0324] The length of the linker spacer sequence and its inverse complementary sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0325] Optionally, 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.

[0326] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0327] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more optionally, it is as shown in SEQ ID NO. 25 or 24.

[0328] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and more optionally, it is read1 or read2 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 26 or 27.

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

[0330] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0331] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0332] In some implementations, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0333] In some implementations, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0334] In some implementations, the linker oligonucleotide is a Y-type linker oligonucleotide, that is, the reverse complementary sequences 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 binding sequence and the downstream sequencing binding sequence are not reverse complementary.

[0335] In some embodiments, the damaged base is any one of a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and baseless site, preferably a U base.

[0336] In some embodiments, the methylated base is, for example, any one of N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymidine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymidine; and the oxidized base is, for example, any one of 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA, and Fapy-dG.

[0337] In some embodiments, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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.

[0338] The thirty-second aspect of this 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 immobilization sequence, an upstream sequencing primer sequence, a linker spacer sequence, a damaged base, and a transposase recognition core sequence, and the second strand comprises the reverse complementary sequence of the transposase recognition core sequence.

[0339] The length of the linker spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0340] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase, and more optionally, it is the ME sequence, and more optionally, it is as shown in SEQ ID NO.23.

[0341] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0342] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and more optionally, it is read1 or read2 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 26 or 27.

[0343] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0344] In some implementations, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0345] In some embodiments, the damaged base is any one of a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and baseless site, preferably a U base.

[0346] In some embodiments, the methylated base is, for example, any one of N3-methyladenine, N7-O6-methylguanine, N3-methylcytosine, and O4-methylthymidine; the alkylated base is, for example, O6-alkylguanine or O4-alkylthymidine; and the oxidized base is, for example, any one of 8-hydroxydeoxyguanosine, 6-methyluracil, thymine glycol, Fapy-dA, and Fapy-dG.

[0347] In some implementations, the first chain further includes an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, the upstream tag sequence 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.

[0348] The thirty-third aspect of this application provides an adaptor oligonucleotide comprising 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 immobilizer sequence, an upstream sequencing primer sequence, and an adaptor spacer sequence, and the second strand comprises, from the 3' end to the 5' end, an optional reverse complementary sequence of a downstream sequencing immobilizer sequence, an reverse complementary sequence of a downstream sequencing primer sequence, an optional reverse complementary sequence of the adaptor spacer sequence, an reverse complementary sequence of an adapter spacer sequence, and an A base.

[0349] 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 aspect twenty-three.

[0350] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0351] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more optionally, it is as shown in SEQ ID NO. 25 or 24.

[0352] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and more optionally, it is read1 or read2 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 26 or 27.

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

[0354] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0355] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0356] In some implementations, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0357] In some implementations, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0358] In some implementations, the adaptor oligonucleotide is a Y-type adaptor oligonucleotide, that is, the reverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary when they are present, and the reverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not reverse complementary when they are present.

[0359] In some embodiments, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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.

[0360] In some embodiments, the length of the adaptor spacer sequence and the reverse complementary sequence of the adaptor spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0361] The thirty-fourth aspect of this application provides an adaptor 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 immobilization sequence, an upstream sequencing primer sequence, and an adaptor spacer sequence, and the second strand comprises, from the 3' end to the 5' end, an optional reverse complementary sequence of a downstream sequencing immobilization sequence, an reverse complementary sequence of a downstream sequencing primer sequence, an reverse complementary sequence of the adaptor spacer sequence, an 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 adaptor oligonucleotide according to the twenty-fourth aspect.

[0362] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0363] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more optionally, it is as shown in SEQ ID NO. 25 or 24.

[0364] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and more optionally, it is read1 or read2 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 26 or 27.

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

[0366] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0367] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0368] In some implementations, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0369] In some implementations, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0370] In some implementations, the adaptor oligonucleotide is a Y-type adaptor oligonucleotide, that is, the reverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary when they are present, and the reverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not reverse complementary when they are present.

[0371] In some embodiments, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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.

[0372] In some embodiments, the length of the adaptor spacer sequence and the reverse complementary sequence of the adaptor spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0373] The thirty-fifth aspect of this application provides an adaptor 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 primer sequence and an adaptor spacer sequence, and the second strand comprises, from the 3' end to the 5' end, an inverse complementary sequence of a downstream sequencing primer sequence, an inverse complementary sequence of the adaptor spacer sequence, an inverse complementary sequence of a linker spacer sequence, and an A base, wherein the inverse complementary sequence of the linker spacer sequence is inversely complementary to the linker spacer sequence of the first strand of the adaptor oligonucleotide according to the twenty-sixth aspect.

[0374] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and more optionally, it is read1 or read2 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 26 or 27.

[0375] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and more optionally, it is read2 or read1 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 27 or 26.

[0376] In some implementations, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0377] In some implementations, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0378] In some implementations, the adaptor oligonucleotide is a Y-type adaptor oligonucleotide, that is, the reverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary.

[0379] In some embodiments, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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.

[0380] In some embodiments, the length of the adaptor spacer sequence and the reverse complementary sequence of the adaptor spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0381] The thirty-sixth aspect of this application provides an adaptor 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 immobilizer sequence, an upstream sequencing primer sequence, and an adaptor spacer sequence, and the second strand comprises, from the 3' end to the 5' end, an inverse complementary sequence of the downstream sequencing immobilizer sequence, an inverse complementary sequence of the downstream sequencing primer sequence, an inverse complementary sequence of the adaptor spacer sequence, an inverse complementary sequence of the adapter spacer sequence, and an A base.

[0382] 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.

[0383] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0384] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more optionally, it is as shown in SEQ ID NO. 25 or 24.

[0385] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and more optionally, it is read1 or read2 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 26 or 27.

[0386] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and more optionally, it is read2 or read1 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 27 or 26.

[0387] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0388] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0389] In some implementations, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0390] In some implementations, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0391] In some implementations, the adaptor oligonucleotide is a Y-type adaptor oligonucleotide, that is, the reverse complementary sequences 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 binding sequence and the downstream sequencing binding sequence are not reverse complementary.

[0392] In some embodiments, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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.

[0393] In some embodiments, the length of the adaptor spacer sequence and the reverse complementary sequence of the adaptor spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0394] The thirty-seventh aspect of this application provides an adaptor oligonucleotide comprising a second strand, wherein the second strand comprises, from the 3' end to the 5' end, an inverse complementary sequence of a downstream sequencing primer sequence, an inverse complementary sequence of an adapter spacer sequence, and an A base.

[0395] 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-ninth aspect.

[0396] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and more optionally, it is read2 or read1 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 27 or 26.

[0397] In some implementations, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0398] In some implementations, 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 aspect twenty-nine.

[0399] In some embodiments, the second strand further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, the downstream tag sequence 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.

[0400] The thirty-eighth aspect of this application provides an adaptor oligonucleotide comprising a second strand, wherein the second strand comprises, from the 3' end to the 5' end, an inverse complementary sequence to a downstream sequencing immobilizer sequence, an inverse complementary sequence to a downstream sequencing primer sequence, an inverse complementary sequence to an adapter spacer sequence, and an A base.

[0401] 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 aspect thirty-two.

[0402] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more optionally, it is as shown in SEQ ID NO. 25 or 24.

[0403] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and more optionally, it is read2 or read1 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 27 or 26.

[0404] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0405] In some implementations, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0406] 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 immobilization sequence is not reverse complementary to the upstream sequencing immobilization sequence of the linker oligonucleotide according to the thirty-second aspect.

[0407] In some embodiments, the second strand further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, the downstream tag sequence 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.

[0408] The thirty-ninth aspect of this application provides a method for constructing a sequencing library, comprising: (1) breaking a target sequence using a transposase complex embedded with an adapter oligonucleotide as described in the twenty-third aspect; (2) treating the sequence with a DNA polymerase and ligase that are intolerant to base damage, optionally adding an adapter oligonucleotide as described in the thirty-third aspect and denaturing and annealing the sequence before ligase treatment; and (3) optionally performing PCR using an extension primer pair.

[0409] The upstream primer of the extension primer pair contains an upstream sequencing immobilization 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 immobilization sequence and the downstream sequencing primer sequence from the 5' end to the 3' end.

[0410] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0411] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more optionally, it is as shown in SEQ ID NO. 25 or 24.

[0412] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0413] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0414] In some implementations, the upstream sequencing binding sequence and the inverse complementary sequence of the downstream sequencing binding sequence are not inversely complementary when present.

[0415] 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.

[0416] In some embodiments, the DNA polymerase that is intolerant to damaged bases is a hypoxanthine-intolerant Pfu DNA polymerase or a Deep vent DNA polymerase.

[0417] 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.

[0418] In some embodiments, the upstream primer further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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' or 3' end of the downstream sequencing primer sequence, 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.

[0419] The fortieth aspect of this application provides a method for constructing a sequencing library, comprising: (1) breaking a target sequence using a transposase complex embedded with an adapter oligonucleotide as described in the twenty-fifth aspect; (2) treating the sequence with a DNA polymerase, FEN1 enzyme, and ligase that are intolerant to base damage, wherein an adapter oligonucleotide as described in the thirty-fifth aspect is added and denatured and annealed prior to ligase treatment; and (3) performing PCR using an extension primer pair.

[0420] The upstream primer of the extension primer pair contains an upstream sequencing immobilization 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 immobilization sequence and the downstream sequencing primer sequence from the 5' end to the 3' end.

[0421] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0422] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more optionally, it is as shown in SEQ ID NO. 25 or 24.

[0423] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0424] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0425] In some implementations, the upstream sequencing binding sequence and the inverse complementary sequence of the downstream sequencing binding sequence are not inversely complementary when present.

[0426] 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.

[0427] In some embodiments, the DNA polymerase that is intolerant to damaged bases is a hypoxanthine-intolerant Pfu DNA polymerase or a Deep vent DNA polymerase.

[0428] 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.

[0429] In some embodiments, the upstream primer further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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' or 3' end of the downstream sequencing primer sequence, 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.

[0430] The forty-first aspect of this application provides a method for constructing a sequencing library, comprising: (1) breaking a target sequence using a transposase complex containing an adapter oligonucleotide as described in the twenty-fifth aspect; and (2) treating the sequence with a DNA polymerase, FEN1 enzyme, and ligase that are intolerant to base damage, wherein the adapter oligonucleotide as described in the thirty-sixth aspect is added and denatured and annealed prior to the ligase treatment.

[0431] 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.

[0432] In some embodiments, the DNA polymerase that is intolerant to damaged bases is a hypoxanthine-intolerant Pfu DNA polymerase or a Deep vent DNA polymerase.

[0433] 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.

[0434] The forty-second aspect of this application provides a method for constructing a sequencing library, comprising: (1) breaking a target sequence using a transposase complex embedded with an adapter oligonucleotide as described in the twenty-sixth aspect; (2) treating the sequence with a DNA polymerase and a ligase intolerant to base damage, wherein an adapter oligonucleotide as described in the thirty-fifth aspect is added prior to the ligase treatment; and (3) performing PCR using an extension primer pair.

[0435] The upstream primer of the extension primer pair contains an upstream sequencing immobilization 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 immobilization sequence and the downstream sequencing primer sequence from the 5' end to the 3' end.

[0436] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0437] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more optionally, it is as shown in SEQ ID NO. 25 or 24.

[0438] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0439] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0440] In some implementations, the upstream sequencing binding sequence and the inverse complementary sequence of the downstream sequencing binding sequence are not inversely complementary when present.

[0441] 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.

[0442] In some embodiments, the DNA polymerase that is intolerant to damaged bases is a hypoxanthine-intolerant Pfu DNA polymerase or a Deep vent DNA polymerase.

[0443] 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.

[0444] In some embodiments, the upstream primer further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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' or 3' end of the downstream sequencing primer sequence, 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.

[0445] The forty-third aspect of this application provides a method for constructing a sequencing library, comprising: (1) breaking a target sequence using a transposase complex containing an adapter oligonucleotide as described in the twenty-sixth aspect; and (2) treating the sequence with a DNA polymerase and a ligase that are intolerant to base damage, wherein an adapter oligonucleotide as described in the thirty-sixth aspect is added prior to the ligase treatment.

[0446] 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.

[0447] In some embodiments, the DNA polymerase that is intolerant to damaged bases is a hypoxanthine-intolerant Pfu DNA polymerase or a Deep vent DNA polymerase.

[0448] 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.

[0449] The forty-fourth aspect of this application provides a method for constructing a sequencing library, comprising: (1) breaking a target sequence using a transposase complex embedded with a linker oligonucleotide as described in the twenty-eighth aspect; (2) treating the sequence with a damage-intolerant DNA polymerase, FEN1 enzyme, and ligase; and (3) performing PCR using an extension primer pair.

[0450] The upstream primer of the extension primer pair contains an upstream sequencing immobilization 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 immobilization sequence and the downstream sequencing primer sequence from the 5' end to the 3' end.

[0451] 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.

[0452] In some embodiments, the DNA polymerase that is intolerant to damaged bases is a hypoxanthine-intolerant Pfu DNA polymerase or a Deep vent DNA polymerase.

[0453] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0454] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more optionally, it is as shown in SEQ ID NO. 25 or 24.

[0455] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0456] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0457] In some implementations, the upstream sequencing binding sequence and the inverse complementary sequence of the downstream sequencing binding sequence are not inversely complementary when present.

[0458] In some embodiments, the upstream primer further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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' or 3' end of the downstream sequencing primer sequence, 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.

[0459] The forty-fifth aspect of this application provides a method for constructing a sequencing library, comprising: (1) breaking a target sequence using a transposase complex embedded with an adapter oligonucleotide as described in the twenty-ninth aspect; (2) treating the sequence with a DNA polymerase and a ligase that are intolerant to base damage, wherein an adapter oligonucleotide as described in the thirty-seventh aspect is added before ligase treatment; and (3) performing PCR using an extension primer pair.

[0460] The upstream primer of the extension primer pair contains an upstream sequencing immobilization 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 immobilization sequence and the downstream sequencing primer sequence from the 5' end to the 3' end.

[0461] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0462] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more optionally, it is as shown in SEQ ID NO. 25 or 24.

[0463] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0464] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0465] In some implementations, the upstream sequencing binding sequence and the inverse complementary sequence of the downstream sequencing binding sequence are not inversely complementary when present.

[0466] 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.

[0467] In some embodiments, the DNA polymerase that is intolerant to damaged bases is a hypoxanthine-intolerant Pfu DNA polymerase or a Deep vent DNA polymerase.

[0468] 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.

[0469] In some embodiments, the upstream primer further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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' or 3' end of the downstream sequencing primer sequence, 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.

[0470] The forty-sixth aspect of this application provides a method for constructing a sequencing library, comprising: (1) breaking a target sequence using a transposase complex containing a linker oligonucleotide as described in the thirty-second aspect; and (2) treating the sequence with a DNA polymerase, FEN1 enzyme, and ligase that are intolerant to base damage.

[0471] 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.

[0472] In some embodiments, the DNA polymerase that is intolerant to damaged bases is a hypoxanthine-intolerant Pfu DNA polymerase or a Deep vent DNA polymerase.

[0473] 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.

[0474] The forty-seventh aspect of this application provides a method for constructing a sequencing library, comprising: (1) breaking a target sequence using a transposase complex containing an adapter oligonucleotide as described in the thirty-second aspect; and (2) treating the sequence with a DNA polymerase and a ligase that are intolerant to base damage, wherein the adapter oligonucleotide as described in the thirty-eighth aspect is added prior to the ligase treatment.

[0475] 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.

[0476] In some embodiments, the DNA polymerase that is intolerant to damaged bases is a hypoxanthine-intolerant Pfu DNA polymerase or a Deep vent DNA polymerase.

[0477] 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.

[0478] The forty-eighth aspect of this application provides a reagent kit comprising...

[0479] (1) The linker oligonucleotide, the integrator oligonucleotide and the extension primer pair as described in aspect 25;

[0480] (2) The linker oligonucleotide according to aspect 25 and the integrator oligonucleotide according to aspect 36;

[0481] (3) The linker oligonucleotides according to aspect 26, the integrator oligonucleotides according to aspect 35, and the extension primer pairs;

[0482] (4) The linker oligonucleotides according to aspect 26 and the integrator oligonucleotides according to aspect 36;

[0483] (5) The linker oligonucleotide and extension primer pair as described in aspect 28;

[0484] (6) The linker oligonucleotide, the integrator oligonucleotide and the extension primer pair as described in aspect 29;

[0485] (7) The linker oligonucleotide as described in aspect 31; or

[0486] (8) The linker oligonucleotides according to aspect 32 and the integrator oligonucleotides according to aspect 38

[0487] The upstream primer of the extension primer pair contains an upstream sequencing immobilization 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 immobilization sequence and the downstream sequencing primer sequence from the 5' end to the 3' end.

[0488] In some embodiments, the upstream primer further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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' or 3' end of the downstream sequencing primer sequence, 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.

[0489] In some embodiments, the kit further comprises one or more of the following: (1) a transposase; (2) a DNA polymerase intolerant to base damage; and (3) a ligase.

[0490] 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.

[0491] In some embodiments, the DNA polymerase that is intolerant to damaged bases is a hypoxanthine-intolerant Pfu DNA polymerase or a Deep vent DNA polymerase.

[0492] 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.

[0493] The forty-ninth aspect of this 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 optional upstream sequencing binding sequence, an optional upstream sequencing primer sequence, an optional linker spacer sequence, one or more restriction sequences, and a transposase recognition core sequence; the second strand comprises, from the 3' end to the 5' end, an optional reverse complementary sequence of a downstream sequencing binding sequence, an optional reverse complementary sequence of a downstream sequencing primer sequence, an optional reverse complementary sequence of the linker spacer sequence, one or more restriction sequences, and an reverse complementary sequence of the transposase recognition core sequence, wherein the restriction sequences and the restriction sequences constitute a recognition / cleavage site for a restriction endonuclease.

[0494] In some embodiments, the transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase, and more optionally, it is the ME sequence, and more optionally, it is as shown in SEQ ID NO.23.

[0495] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0496] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more optionally, it is as shown in SEQ ID NO. 25 or 24.

[0497] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and more optionally, it is read1 or read2 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 26 or 27.

[0498] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and more optionally, it is read2 or read1 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 27 or 26.

[0499] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0500] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0501] In some implementations, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0502] In some implementations, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0503] In some implementations, the linker oligonucleotide is a Y-type linker oligonucleotide, that is, the reverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary when they are present, and the reverse complementary sequences of the upstream sequencing binding sequence and the downstream sequencing binding sequence are not reverse complementary when they are present.

[0504] In some embodiments, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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.

[0505] In some embodiments, the length of the linker spacer sequence and its inverse complementary sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0506] In some embodiments, the restriction endonuclease is a restriction endonuclease known to those skilled in the art to recognize restriction sequences and cleave 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.

[0507] In some embodiments, the adhesive end is a 5' adhesive end or a 3' adhesive end.

[0508] In some embodiments, the sticky end has up to 2, 3, 4, 5, 6, 7, 8, 9 or 10 protruding unpaired bases.

[0509] In some implementations, the linker oligonucleotide may have a linker spacer sequence added to the 5' end of the restriction site to improve cleavage efficiency.

[0510] A fiftieth aspect of this application provides an adaptor oligonucleotide comprising a first strand and a second strand, wherein the first strand comprises, from its 5' end to its 3' end, an optional upstream sequencing immobilizer sequence, an upstream sequencing primer sequence, an optional adaptor spacer sequence, an optional adapter spacer sequence, and a restriction sequence three; and the second strand comprises, from its 3' end to its 5' end, an optional reverse complementary sequence of a downstream sequencing immobilizer sequence, an 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 adapter spacer sequence, and a restriction sequence four.

[0511] The joint spacer sequence is the same as that described in aspect 49.

[0512] Wherein, restriction sequence three and restriction sequence four constitute the post-cleavage sticky end of the restriction endonuclease described in aspect forty-nine.

[0513] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0514] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more optionally, it is as shown in SEQ ID NO. 25 or 24.

[0515] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and more optionally, it is read1 or read2 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 26 or 27.

[0516] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and more optionally, it is read2 or read1 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 27 or 26.

[0517] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0518] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0519] In some implementations, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0520] In some implementations, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0521] In some implementations, the adaptor oligonucleotide is a Y-type adaptor oligonucleotide, that is, the reverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary when they are present, and the reverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not reverse complementary when they are present.

[0522] In some embodiments, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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.

[0523] In some embodiments, the length of the adaptor spacer sequence and the reverse complementary sequence of the adaptor spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0524] The fifty-first aspect of this application provides an adaptor 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 primer sequence, an optional adaptor spacer sequence, an optional adapter spacer sequence, and a restriction sequence three; and the second strand comprises, from the 3' end to the 5' end, an inverse complementary sequence of a downstream sequencing primer sequence, an optional inverse complementary sequence of the adaptor spacer sequence, an optional inverse complementary sequence of the adapter spacer sequence, and a restriction sequence four.

[0525] The joint spacer sequence is the same as that described in aspect 49.

[0526] Wherein, restriction sequence three and restriction sequence four constitute the post-cleavage sticky end of the restriction endonuclease described in aspect forty-nine.

[0527] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and more optionally, it is read1 or read2 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 26 or 27.

[0528] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and more optionally, it is read2 or read1 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 27 or 26.

[0529] In some implementations, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0530] In some implementations, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0531] In some implementations, the adaptor oligonucleotide is a Y-type adaptor oligonucleotide, that is, the reverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary.

[0532] In some embodiments, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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.

[0533] In some embodiments, the length of the adaptor spacer sequence and the reverse complementary sequence of the adaptor spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0534] The fifty-second aspect of this application provides an adaptor 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 immobilizer sequence, an upstream sequencing primer sequence, an optional adaptor spacer sequence, an optional adapter spacer sequence, and a restriction sequence three; and the second strand comprises, from the 3' end to the 5' end, an inverse complementary sequence of the downstream sequencing immobilizer sequence, an inverse complementary sequence of the downstream sequencing primer sequence, an inverse complementary sequence of the optional adaptor spacer sequence, an inverse complementary sequence of the optional adapter spacer sequence, and a restriction sequence four.

[0535] The joint spacer sequence is the same as that described in aspect 49.

[0536] Wherein, restriction sequence three and restriction sequence four constitute the post-cleavage sticky end of the restriction endonuclease described in aspect forty-nine.

[0537] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0538] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more optionally, it is as shown in SEQ ID NO. 25 or 24.

[0539] In some embodiments, the upstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and more optionally, it is read1 or read2 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 26 or 27.

[0540] In some embodiments, the downstream sequencing primer sequence is a sequencing primer sequence of the Illumina platform, and more optionally, it is read2 or read1 of the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 27 or 26.

[0541] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0542] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0543] In some implementations, the upstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0544] In some implementations, the downstream sequencing primer sequence is a sequencing primer sequence of the MGI platform, and more optionally, it is a one-stranded sequencing primer or a two-stranded sequencing primer of the MGI platform, and more optionally, it is Read1 or Read2 of the MGI platform.

[0545] In some implementations, the adaptor oligonucleotide is a Y-type adaptor oligonucleotide, that is, the reverse complementary sequences 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 binding sequence and the downstream sequencing binding sequence are not reverse complementary.

[0546] In some embodiments, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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.

[0547] In some embodiments, the length of the adaptor spacer sequence and the reverse complementary sequence of the adaptor spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

[0548] The fifty-third aspect of this application provides a method for constructing a sequencing library, comprising: (1) breaking a target sequence using a transposase complex containing an adapter oligonucleotide as described in the forty-ninth aspect; (2) treating the target sequence with DNA polymerase, the restriction endonuclease as described in the forty-ninth aspect, and a ligase, optionally adding an adapter oligonucleotide as described in the fiftieth aspect and denaturing and annealing the target sequence before ligase treatment; and (3) optionally performing PCR using an extension primer pair.

[0549] The upstream primer of the extension primer pair contains an upstream sequencing immobilization 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 immobilization sequence and the downstream sequencing primer sequence from the 5' end to the 3' end.

[0550] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0551] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more optionally, it is as shown in SEQ ID NO. 25 or 24.

[0552] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0553] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0554] In some implementations, the upstream sequencing binding sequence and the inverse complementary sequence of the downstream sequencing binding sequence are not inversely complementary when present.

[0555] In some embodiments, the upstream primer further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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' or 3' end of the downstream sequencing primer sequence, 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.

[0556] The fifty-fourth aspect of this application discloses a method for constructing a sequencing library, comprising: (1) breaking a target sequence using a transposase complex containing an adapter oligonucleotide as described in the forty-ninth aspect; (2) treating the target sequence with DNA polymerase, the restriction endonuclease as described in the forty-ninth aspect, and a ligase, wherein an adapter oligonucleotide as described in the fifty-first aspect is added before ligase treatment; and (3) performing PCR using an extension primer pair.

[0557] The upstream primer of the extension primer pair contains an upstream sequencing immobilization 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 immobilization sequence and the downstream sequencing primer sequence from the 5' end to the 3' end.

[0558] In some implementations, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform, and more optionally, it is a P5 or P7 sequence from the Illumina platform, and more optionally, it is as shown in SEQ ID NO. 24 or 25.

[0559] In some embodiments, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the Illumina platform, more optionally, it is the Illumina platform P7 or P5, and more optionally, it is as shown in SEQ ID NO. 25 or 24.

[0560] In some implementations, the upstream sequencing-fixed sequence is a sequencing-fixed sequence of the MGI platform, and more optionally, it is an upstream or downstream clamping connector sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle connector or a vesicular sequence of a vesicle connector of the MGI platform.

[0561] In some implementations, the downstream sequencing immobilization sequence is a sequencing immobilization sequence of the MGI platform, and more optionally, it is an upstream or downstream splint adapter sequence of the MGI platform, and more optionally, it is a linear sequence of a vesicle adapter or a vesicular sequence of a vesicle adapter of the MGI platform.

[0562] In some implementations, the upstream sequencing binding sequence and the inverse complementary sequence of the downstream sequencing binding sequence are not inversely complementary when present.

[0563] In some embodiments, the upstream primer further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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' or 3' end of the downstream sequencing primer sequence, 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.

[0564] The fifty-fifth aspect of this application provides a method for constructing a sequencing library, comprising: (1) breaking a target sequence using a transposase complex containing an adapter oligonucleotide as described in the forty-ninth aspect; and (2) treating the sequence with a DNA polymerase, a restriction endonuclease as described in the forty-ninth aspect, and a ligase, wherein the adapter oligonucleotide as described in the fifty-second aspect is added prior to the ligase treatment.

[0565] The fifty-sixth aspect of this application is a reagent kit comprising...

[0566] (1) The linker oligonucleotide, the integrator oligonucleotide, and the extension primer pair as described in aspect 49; or

[0567] (2) The linker oligonucleotides according to aspect 49 and the integrator oligonucleotides according to aspect 52

[0568] The upstream primer of the extension primer pair contains an upstream sequencing immobilization 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 immobilization sequence and the downstream sequencing primer sequence from the 5' end to the 3' end.

[0569] In some embodiments, the upstream primer further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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' or 3' end of the downstream sequencing primer sequence, 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.

[0570] In some embodiments, the kit further comprises one or more of the following: (1) a transposase; (2) a DNA polymerase; (3) the restriction endonuclease described in aspect 49; and (4) a ligase.

[0571] In some embodiments, the DNA polymerase is any conventional DNA polymerase known to those skilled in the art that can be used for PCR, including but not limited to Taq DNA polymerase, DNA polymerase I-V, Tth DNA polymerase, Pfu DNA polymerase, E. coli DNA polymerase I, Bst DNA polymerase, T7 DNA polymerase, Vent DNA polymerase, and KOD DNA polymerase. Attached Figure Description

[0572] Figure 1: The principle of fragmented enzymatic library construction and the principle of conventional transposable enzymatic library construction;

[0573] Figure 2: Schematic diagram of the structure of the Tn5-Y complex;

[0574] Figure 3: Schematic diagram of the structure of the Tn5-YU complex;

[0575] Figure 4: Schematic diagram of the structure of the Tn5-oligo complex;

[0576] Figure 5: Schematic diagram of the structure of the Tn5-oligo-PCR-free complex;

[0577] Figure 6: Schematic diagram of the structure of the Tn5-oligo-Y complex;

[0578] Figure 7: Schematic diagram of the structure of the Tn5-CUT complex;

[0579] Figure 8: Flowchart of library construction for the Tn5-Y complex;

[0580] Figure 9: Flowchart of library construction for the Tn5-YU complex;

[0581] Figure 10: Library outputs obtained by fragmentation enzymatic method, conventional transposase method, Tn5-Y complex and Tn5-YU complex respectively;

[0582] Figure 11A: Peak diagrams of library preparations obtained by fragmented enzyme method, conventional transposase method, Tn5-Y complex and Tn5-YU complex at an input of 100 pg.

[0583] Figure 11B: Peak diagrams of library preparations obtained by fragmented enzymatic method, conventional transposase method, Tn5-Y complex and Tn5-YU complex at an input of 1 ng.

[0584] Figure 11C: Peak diagrams of library preparations obtained by fragmented enzyme method, conventional transposase method, Tn5-Y complex and Tn5-YU complex at an input of 10 ng.

[0585] Figure 12: Flowchart of library construction for the Tn5-oligo complex;

[0586] Figure 13: Flowchart of library construction for the Tn5-oligo-PCR-free complex;

[0587] Figure 14: Flowchart of routine library preparation for the Tn5-oligo-Y complex;

[0588] Figure 15: PCR-free library preparation flowchart of Tn5-oligo-Y complex;

[0589] Figure 16: Flowchart of routine library preparation for the Tn5-CUT complex;

[0590] Figure 17: PCR-free library preparation flowchart for the Tn5-CUT complex;

[0591] Figure 18: Comparison of library output from conventional transposase library construction, Tn5-oligo complex library construction, conventional Tn5-oligo-Y complex library construction, and Tn5-CUT complex library construction.

[0592] Figure 19: Comparison of library peak diagrams for conventional transposase library construction, Tn5-oligo complex library construction, conventional Tn5-oligo-Y complex library construction, and Tn5-CUT complex library construction.

[0593] Figure 20: Library outputs obtained by conventional transposase library construction, Tn5-oligo complex library construction, and linker replacement strategy library construction;

[0594] Figure 21: Ligation efficiency between oligonucleotide linkers and adaptors containing oligos of different lengths;

[0595] Figure 22: Comparison of library output from conventional transposase library construction, Tn5-YU complex library construction, Tn5-Y-U1 complex library construction, Tn5-Y-U2 complex library construction, and Tn5-Y-U3 complex library construction;

[0596] Figure 23: Comparison of library peak diagrams for conventional transposase library construction, Tn5-YU complex library construction, Tn5-Y-U1 complex library construction, Tn5-Y-U2 complex library construction, and Tn5-Y-U3 complex library construction;

[0597] Figure 24: Comparison of library output from conventional transposase library construction, Tn5-oligo complex library construction, Tn5-oligo1 complex library construction, and Tn5-oligo2 complex library construction;

[0598] Figure 25: Comparison of library peak diagrams for conventional transposase library construction, Tn5-oligo complex library construction, Tn5-oligo1 complex library construction, and Tn5-oligo2 complex library construction.

[0599] Detailed Implementation Methods (Examples)

[0600] The technical solution of this application will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of this application and do not represent or limit the scope of protection of this application. The scope of protection of this application shall be determined by the claims.

[0601] In the following embodiments, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the art; unless otherwise specified, all experimental methods and techniques used were conventional methods and techniques in the art.

[0602] Example 1

[0603] Preparation of different Tn5 transposase complexes

[0604] Oligo nucleotides Oligo 1 and Oligo 2 were synthesized at Sangon Biotech (Shanghai). Oligo 1 includes SEQ ID NO. 1-6, and Oligo 2 includes SEQ ID NO. 7-9. The specific sequences are as follows:

[0605] Table 1

[0606] Dissolve Oligo 1 and Oligo 2 separately in PBS to 10 μM. Take 10 μl of Oligo 1 + 10 μl of Oligo 2 to form reaction 1. Vortex reaction 1 thoroughly to mix, and briefly centrifuge to return the solution to the bottom of the tube. Place the tube in a PCR instrument and proceed with the following reaction program:

[0607] Table 2

[0608] After the reaction was completed, the product of reaction 1 was named Tn5 Adapter.

[0609] The following reaction components were then added sequentially to the sterile PCR tube:

[0610] Table 3

[0611] Note: Tn5 in Table 2 was obtained by expression using the psfTn5 plasmid (Addgene plasmid#79107).

[0612] Use a pipette to gently pipette 20 times to mix thoroughly, and place at 30°C for 1 hour. The reaction product is named the Tn5 complex and stored at -30 to -15°C.

[0613] The Oligo A sequences are: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6.

[0614] The Oligo B sequences are: SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9.

[0615] SEQ ID NO.1 and SEQ ID NO.7 are used to form the Tn5-Y complex, as shown in Figure 2; SEQ ID NO.2 and SEQ ID NO.7 are used to form the Tn5-YU complex, as shown in Figure 3; SEQ ID NO.8 and SEQ ID NO.3 are used to form the Tn5-oligo complex, as shown in Figure 4; SEQ ID NO.8 and SEQ ID NO.4 are used to form the Tn5-oligo-PCR-free complex, as shown in Figure 5; SEQ ID NO.8 and SEQ ID NO.5 are used to form the Tn5-oligo-Y complex, as shown in Figure 6; and SEQ ID NO.6 and SEQ ID NO.9 are used to form the Tn5-CUT complex, as shown in Figure 7.

[0616] Example 2

[0617] This embodiment uses the gDNA genome of 293 cells as an example template and compares experiments using the methods of "construction of library with conventional Tn5 products", "fragmented enzymatic library construction", "Tn5-Y complex library construction" and "Tn5-YU complex library construction" to illustrate the method steps and beneficial effects of this application.

[0618] 1. Database construction for standard Tn5 products (using Novizan product TD502)

[0619] 100 pg, 1 ng, and 10 ng of purified 293 cell gDNA were used as starting DNA templates, and library preparation was performed according to “09 / Experimental Procedure” in the Vazyme#TD502 product manual.

[0620] Second-generation sequencing

[0621] The library was sent to Nanjing Shihe Gene Biotechnology Co., Ltd. for sequencing using a HiseqX sequencing instrument.

[0622] 2. Fragmented enzymatic library construction (using Novizan product ND617)

[0623] 100 pg, 1 ng, and 10 ng of purified 293 cell gDNA were used as starting DNA templates, respectively, and libraries were prepared according to the "08 / Standard Experimental Protocol" in the Vazyme#ND617 product manual.

[0624] Second-generation sequencing

[0625] The second-generation sequencing method is the same as the conventional Tn5 product library construction method described above.

[0626] 3. Tn5-Y complex library construction (using some reagents from Novizan product TD502)

[0627] DNA fragmentation

[0628] Thaw 5×TTBL (Vazyme#TD502) at room temperature, invert and mix well before use. Confirm that 5×TS (Vazyme#TD502) is at room temperature and gently tap the tube wall to check for any precipitate. If precipitate is present, heat at 37°C and vortex to mix; the precipitate will dissolve.

[0629] The following reaction system was prepared in a sterile PCR tube, containing 100 pg, 1 ng, and 10 ng of purified 293 cell gDNA as the starting DNA template:

[0630] Table 4

[0631] Use a pipette to gently blow and mix 20 times to ensure thorough mixing.

[0632] Place the reaction tube in the PCR instrument and run the following reaction program:

[0633] Table 5

[0634] DNA fragment repair linker

[0635] Immediately after the reaction was complete, add 5 μl of 5×TS to the product, gently pipette to mix thoroughly, and incubate at room temperature for 5 minutes. Then, immediately prepare the repair ligation system and proceed with the reaction. The specific system and reaction are as follows:

[0636] Table 6

[0637] NEBuffer 2: 50mM NaCl, 10mM Tris-HCl, 10mM MgCl2, 1mM DTT, pH 7.925℃, the same applies below.

[0638] After mixing thoroughly by blowing, proceed with the following reaction:

[0639] Table 7

[0640] Repairing ligation product purification

[0641] Add 60 μl of magnetic beads (Nanjing Novizan product N411) to the PCR reaction product above. Vortex or pipette 10 times to ensure homogeneity. Incubate at room temperature for 5 min. Briefly centrifuge the reaction tube and place it on a magnetic rack to separate the magnetic beads and liquid. After the solution becomes clear (about 5 min), carefully remove the supernatant, being careful not to disturb the magnetic beads. Keep the centrifuge tube on the magnetic rack and add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads. Incubate at room temperature for 30 sec, and carefully remove the supernatant. Repeat this step, rinsing twice in total. Keep the centrifuge tube on the magnetic rack and air dry for 3-5 min. After the magnetic beads have dried, remove the centrifuge tube from the magnetic rack, add 22 μl of sterile ultrapure water to elute, vortex or pipette 10 times to thoroughly mix the magnetic beads, and incubate at room temperature for 5 min. Briefly centrifuge the PCR tube and collect the contents. Place the tube on a magnetic rack to separate the magnetic beads and liquid. After the solution has clarified (about 5 minutes), carefully aspirate 20 μl of the supernatant and transfer it to a new EP tube. Store at -30 to -15°C.

[0642] PCR enrichment

[0643] Immediately after the reaction is complete, add 5 μl of 5×TS (Vazyme#TD502) to the product, gently mix with a pipette, and incubate 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:

[0644] Table 8

[0645] *8 types of N5XX and 12 types of N7XX are from Nanjing Novizan's TD202 product.

[0646] After the above system is mixed by blowing, the following reaction is carried out:

[0647] Table 9

[0648] PCR product purification

[0649] Vortex to mix the DNA purification magnetic beads (Nanjing Novizan product N411) and add 25 μl of the magnetic beads to the PCR reaction product above. Vortex or pipette 10 times to ensure homogeneity and incubate at room temperature for 5 min. Briefly centrifuge the reaction tube and place it on a magnetic rack to separate the magnetic beads and liquid. After the solution becomes clear (about 5 min), carefully transfer the supernatant to a new centrifuge tube, being careful not to disturb the magnetic beads. Add 7.5 μl of the magnetic beads to the supernatant and vortex or pipette 10 times to ensure homogeneity. Incubate at room temperature for 5 min. Place the tube on a magnetic rack and wait for the solution to become clear, then remove the supernatant. Keeping the centrifuge tube on the magnetic rack, add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads and incubate at room temperature for 30 sec. Carefully remove the supernatant. Repeat this step, rinsing twice in total. Keeping the centrifuge tube on the magnetic rack, open the cap and air dry for 3-5 min. After the magnetic beads have dried, remove the centrifuge tube from the magnetic rack, add 22 μl of sterile ultrapure water to wash, vortex or pipette 10 times to thoroughly mix the magnetic beads, and incubate at room temperature for 5 min. Briefly centrifuge the PCR tube to collect the collected magnetic beads and liquid, and place it on the magnetic rack to separate the magnetic beads and liquid. After the solution has clarified (about 5 min), carefully aspirate 20 μl of the supernatant and transfer it to a new EP tube, and store at -30 to -15℃.

[0650] Document Quality Inspection

[0651] The prepared library was tested for length distribution on an Agilent 2100 Bioanalyzer.

[0652] Second-generation sequencing

[0653] The same method as the conventional Tn5 product database construction method described above.

[0654] 4. Tn5-YU complex library construction (using some reagents from Novizan product TD502)

[0655] DNA fragmentation

[0656] Thaw 5×TTBL (Vazyme#TD502) at room temperature, invert and mix well before use. Confirm that 5×TS (Vazyme#TD502) is at room temperature and gently tap the tube wall to check for any precipitate. If precipitate is present, heat at 37°C and vortex to mix; the precipitate will dissolve.

[0657] The following reaction system was prepared in a sterile PCR tube, wherein 100 pg, 1 ng, and 10 ng of purified 293 cell gDNA were used as the starting DNA template:

[0658] Table 10

[0659] Use a pipette to gently blow and mix 20 times to ensure thorough mixing.

[0660] Place the reaction tube in the PCR instrument and run the following reaction program:

[0661] Table 11

[0662] DNA fragment repair linker

[0663] Immediately after the reaction was complete, add 5 μl of 5×TS (Vazyme#TD502) to the product, gently pipette to mix thoroughly, and incubate at room temperature for 5 min. Then, immediately prepare the repair ligation system and proceed with the reaction. The specific system and reaction are as follows:

[0664] Table 12

[0665] *FEN1 was obtained by the method disclosed in the reference: Bornarth, CJ, Ranalli, TA, Henricksen, LA, Wahl, AF, and Bambara, RA (1999) Biochemistry 38, 13347-13354.

[0666] After mixing thoroughly by blowing, proceed with the following reaction:

[0667] Table 13

[0668] Repairing ligation product purification

[0669] Add 60 μl of magnetic beads (Nanjing Novizan product N411) to the PCR reaction product above. Vortex or pipette 10 times to ensure homogeneity. Incubate at room temperature for 5 min. Briefly centrifuge the reaction tube and place it on a magnetic rack to separate the magnetic beads and liquid. After the solution becomes clear (about 5 min), carefully remove the supernatant, being careful not to disturb the magnetic beads. Keep the centrifuge tube on the magnetic rack and add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads. Incubate at room temperature for 30 sec, and carefully remove the supernatant. Repeat this step, rinsing twice in total. Keep the centrifuge tube on the magnetic rack and air dry for 3-5 min. After the magnetic beads have dried, remove the centrifuge tube from the magnetic rack, add 22 μl of sterile ultrapure water to elute, vortex or pipette 10 times to thoroughly mix the magnetic beads, and incubate at room temperature for 5 min. Briefly centrifuge the PCR tube and collect the contents. Place the tube on a magnetic rack to separate the magnetic beads and liquid. After the solution has clarified (about 5 minutes), carefully aspirate 20 μl of the supernatant and transfer it to a new EP tube. Store at -30 to -15°C.

[0670] The specific steps for PCR enrichment, PCR product purification, library quality testing, and next-generation sequencing are the same as those for the Tn5-Y complex library construction method described above.

[0671] The resulting library output is shown in Figure 10. The peak diagrams of the library are shown in Figures 11A, 11B, and 11C, respectively. The sequencing analysis is shown in Table 14.

[0672] Table 14

[0673] Experimental results

[0674] As shown in Figure 10, the library yields of Tn5-Y and Tn5-YU complexes were significantly better than those of libraries prepared with conventional Tn5 at different starting amounts. In particular, the library yield of Tn5-YU complex exceeded that of Tn5-Y complex at all input amounts, indicating that adding U base modification to the Y-linker embedded with transposase can overcome the library loss caused by polymerase chain displacement activity.

[0675] As shown in Figures 11A, 11B, and 11C, the library distribution shows that, under different input levels, the Tn5-Y and Tn5-YU complexes have the same size distribution as the conventional Tn5 library fragments, and both can effectively disrupt the genome.

[0676] Sequencing analysis results are shown in Table 14. Uniformity values ​​indicate that Tn5-YU improves library uniformity by approximately one-fold compared to conventional Tn5, and significantly reduces the required sequencing depth (requiring a depth of 10X or higher). Uniformity refers to the uniformity of the distribution of read data across the genome or target region; better uniformity results in a smaller value. There were no differences in conventional metrics such as mapping rate (the percentage of reads that align with the reference genome), coverage (the extent to which the spliced ​​reads cover the reference genome; higher is better), and softclip (reads matching different regions of the reference genome).

[0677] Example 3

[0678] In this embodiment, 10 ng of 293 cell gDNA was used as a DNA template, and the following were used:

[0679] ① Database construction for standard Tn5 products;

[0680] ② Tn5-oligo complex library construction method (flow shown in Figure 12), the oligo region in the Tn5-oligo complex is 12nt (indicated by underscore in SEQ ID NO.3), and the Tn5-oligo linker used for connection is SEQ ID NO.10;

[0681] ③ The method for constructing a library of the Tn5-oligo-PCR-free complex (the flowchart is shown in Figure 13). The oligo region in the Tn5-oligo-Y complex is 12nt (underlined in SEQ ID NO.4). The Tn5-oligo-PCR-free linker used for ligation is SEQ ID NO.11.

[0682] ④: Conventional library construction method for Tn5-oligo-Y complex (flow shown in Figure 14). The oligo region in the Tn5-oligo-Y complex is 6nt (indicated by underscore in SEQ ID NO.5). The Tn5-oligo-Y linker used for connection is formed by annealing SEQ ID NO.10 and SEQ ID NO.13.

[0683] ⑤: PCR-free library preparation method for Tn5-oligo-Y complex (flow chart shown in Figure 15). The oligo region in Tn5-oligo-Y complex is 6nt (underlined in SEQ ID NO.5). The Tn5-oligo-Y-PCR-free-adaptor used for ligation is formed by annealing SEQ ID NO.12 and SEQ ID NO.14.

[0684] ⑥: Library construction method of Tn5-oligo-CUT complex (flowchart shown in Figure 16). The Tn5-oligo-CUT complex contains a sticky end recognition region (underlined in SEQ ID NO. 6). The Tn5-oligo-adaptor used for ligation is formed by annealing SEQ ID NO. 13 and SEQ ID NO. 15. (Similarly, the adaptor can also be used to achieve PCR-free library construction of Tn5-CUT complex, flowchart shown in Figure 17).

[0685] The following table lists the connectants involved in the aforementioned sections, which were compared experimentally:

[0686] Table 15

[0687] The specific steps are as follows:

[0688] ①: The database construction for conventional Tn5 products follows the same steps as in Example 2 above.

[0689] ②: Tn5-oligo complex library construction (using some reagents from Vazyme#TD502)

[0690] DNA fragmentation

[0691] Thaw 5×TTBL (Vazyme#TD502) at room temperature, invert and mix well before use. Confirm that 5×TS (Vazyme#TD502) is at room temperature and gently tap the tube wall to check for any precipitate. If precipitate is present, heat at 37°C and vortex to mix; the precipitate will dissolve.

[0692] Prepare the following reaction system in a sterile PCR tube:

[0693] Table 16

[0694] Use a pipette to gently blow and mix 20 times to ensure thorough mixing.

[0695] Place the reaction tube in the PCR instrument and run the following reaction program:

[0696] Table 17

[0697] DNA fragment repair linker

[0698] Sequence 8 was diluted to 10 μM with sterile water and set aside. Immediately after the reaction was complete, 5 μl of 5×TS was added to the product, and the mixture was gently pipetted to mix thoroughly. After incubating at room temperature for 5 min, the repair ligation system was immediately prepared and the reaction was carried out. The specific system and reaction are as follows:

[0699] Table 18

[0700] After mixing thoroughly by blowing, proceed with the following reaction:

[0701] Table 19

[0702] The specific steps for repairing ligation product purification, PCR enrichment, PCR product purification, library quality testing, and next-generation sequencing are the same.

[0703] Example 2.

[0704] ③: Library construction of Tn5-oligo-PCR-free complex (using some reagents from Vazyme#TD502)

[0705] This method does not require a PCR enrichment step. The DNA fragmentation uses the Tn5-oligo-PCR-free complex prepared in Example 1, and the DNA fragment repair ligation uses the Tn5-oligo-PCR-free adapter (SEQ ID NO.11). The remaining steps are the same as those described in ② Tn5-oligo complex library construction above.

[0706] ④: Conventional library construction method for Tn5-oligo-Y complex (using some reagents from Vazyme#TD502)

[0707] DNA fragmentation was performed using the Tn5-oligo-Y complex prepared in Example 1. DNA fragment repair ligation was performed using the Tn5-oligo-Y-adaptor, which was annealed from SEQ ID NO.10 and SEQ ID NO.13. The specific steps are as follows:

[0708] Dilute SEQ ID NO.10 and SEQ ID NO.13 with sterile water to 10 μM for later use. Take 10 μl of SEQ ID NO.10 and 10 μl of SEQ ID NO.13, mix them, vortex to mix thoroughly, and briefly centrifuge to return the solution to the bottom of the tube. Place the tube in a PCR instrument and perform the following reaction program: incubate at 95℃ for 2 min, then gradually decrease the temperature to 25℃ at a gradient of 0.2℃ per second. After the reaction is complete, the reaction product, Tn5-oligo-Y-adaptor, is obtained and stored on ice for later use.

[0709] The amount of Tn5-oligo-Y-adaptor used in the DNA fragment repair ligation step is 5 μl, and the amount of pure water is adjusted to keep the total volume of the reaction system constant. The remaining steps are the same as those described in ② Tn5-oligo complex library construction above.

[0710] ⑤: PCR-free library preparation method for Tn5-oligo-Y complex (using some reagents from Vazyme#TD502)

[0711] This method does not require a PCR enrichment step. DNA fragmentation uses the Tn5-oligo-Y complex prepared in Example 1. DNA fragment repair and ligation uses the Tn5-oligo-Y-PCR-free-adaptor, which is formed by annealing SEQ ID NO.12 and SEQ ID NO.14. The annealing method is the same as above. The remaining steps are the same as the conventional library construction method of the Tn5-oligo-Y complex in ④ above.

[0712] ⑥: Tn5-oligo-CUT complex library construction method (using some reagents from Vazyme#TD502)

[0713] DNA fragmentation

[0714] Thaw 5×TTBL (Vazyme#TD502) at room temperature, invert and mix well before use. Confirm that 5×TS (Vazyme#TD502) is at room temperature and gently tap the tube wall to check for any precipitate. If precipitate is present, heat at 37°C and vortex to mix; the precipitate will dissolve.

[0715] Prepare the following reaction system in a sterile PCR tube:

[0716] Table 20

[0717] Use a pipette to gently blow and mix 20 times to ensure thorough mixing.

[0718] Place the reaction tube in the PCR instrument and run the following reaction program:

[0719] Table 21

[0720] DNA fragment repair

[0721] Immediately after the fragmentation reaction was complete, add 5 μl of 5×TS to the product, gently pipette to mix thoroughly, and incubate at room temperature for 5 min. Then immediately prepare the repair ligation system and proceed with the reaction. The specific system and reaction are as follows:

[0722] Table 22

[0723] NEB Buffer 2: 50mM NaCl, 10mM Tris-HCl, 10mM MgCl2, 1mM DTT, pH7.9.

[0724] Purification of repair products

[0725] Purification was performed using 50 μl of magnetic beads (Nanjing Novizan product N411), following the same procedure as above.

[0726] restriction enzyme digestion

[0727] Prepare the restriction enzyme digestion system and carry out the reaction. The specific system and reaction are as follows:

[0728] Table 23

[0729] 10×NEBuffer r3.1: 1M NaCl, 500mM Tris-HCl, 100mM MgCl2, 1mg / ml Recombinant Albumin (pH 7.9)

[0730] After mixing thoroughly by blowing, proceed with the following reaction:

[0731] Table 24

[0732] DNA fragment restriction enzyme digestion product ligation

[0733] The Tn5-CUT-adaptor is formed by annealing sequences SEQ ID NO.13 and SEQ ID NO.15: Sequences SEQ ID NO.13 and SEQ ID NO.15 are diluted to 10 μM with sterile water. 10 μl of sequence SEQ ID NO.13 and 10 μl of sequence SEQ ID NO.15 are mixed and vortexed thoroughly. The mixture is then briefly centrifuged to return the solution to the bottom of the tube. The tube is placed in a PCR instrument and the following reaction program is performed: incubation at 95°C for 2 min, followed by a gradient cooling to 25°C at 0.2°C per second. After the reaction is complete, the reaction product, the Tn5-CUT-adaptor, is obtained and stored on ice for later use.

[0734] After the restriction enzyme digestion reaction was completed, the repair ligation system was prepared and the reaction was carried out. The specific system and reaction are as follows:

[0735] Table 25

[0736] After mixing thoroughly by blowing, proceed with the following reaction:

[0737] Table 26

[0738] Ligation product purification

[0739] Purification was performed using 60 μl of magnetic beads (Nanjing Novizan product N411), following the same steps as above.

[0740] The steps for PCR enrichment, PCR product purification, library quality testing, and next-generation sequencing are the same as above.

[0741] The library output is shown in Figure 18, and the library peak diagram is shown in Figure 19. For the Tn5-oligo-PCR-free and Tn5-oligo-γ-PCR-free complex libraries, since PCR amplification is not required, all products were sent for sequencing and are not included in the library output and peak diagram statistics. All sequencing data are shown in Table 27.

[0742] Table 27

[0743] Experimental results

[0744] As shown in Figure 18, the library output of Tn5-oligo complex, conventional Tn5-oligo-Y complex, and Tn5-CUT complex is significantly better than that of conventional Tn5 library preparation. Specifically, the library output of the three test groups is more than three times higher than that of the conventional Tn5 library preparation group.

[0745] As shown in Figure 19, the library distribution shows that the Tn5-oligo complex library, the conventional Tn5-oligo-Y complex library, and the Tn5-CUT complex library have the same fragment size distribution as the conventional Tn5 library, and all of them can effectively disrupt the genome.

[0746] The sequencing analysis results are shown in Table 27. The Uniformity values ​​show that Tn5-oligo complex library preparation, Tn5-oligo-PCR-free complex library preparation, conventional Tn5-oligo-Y complex library preparation, PCR-free Tn5-oligo-Y complex library preparation, and Tn5-CUT complex library preparation improved library uniformity compared to conventional Tn5 library preparation and significantly reduced the sequencing depth requirement (requiring a depth of 10X or higher). There were no differences between the test groups and conventional Tn5 library preparation in terms of mapping rate, coverage, and softclip.

[0747] Example 4

[0748] This embodiment uses the gDNA genome of 293 cells as an example template and compares the experimental steps and beneficial effects of the present application using the "conventional Tn5 product library construction method", the "Tn5-oligo complex library construction method" and the "adapter replacement strategy disclosed in Chinese Patent CN115552035A".

[0749] 1. The conventional method for building a database for Tn5 products is the same as in Example 2.

[0750] 2. The method for constructing the Tn5-oligo complex library is the same as in Example 3.

[0751] 3. The connector replacement strategy disclosed in Chinese Patent CN115552035A is carried out according to the steps in the aforementioned patent specification.

[0752] The statistical library yield is shown in Figure 20. The Tn5-oligo complex library yield is better than that of the linker replacement strategy. Both are better than the library yield prepared by conventional Tn5. The linker replacement strategy theoretically avoids the library loss caused by the uncontrollable chain replacement process mentioned above. However, the overall library preparation process involves multiple opening of the cap to add reagents and mix, which is complicated, time-consuming and results in a large loss of library output. The method of this application can avoid the above shortcomings.

[0753] Example 5

[0754] Based on the aforementioned Tn5-oligo-Y complex, the lengths of the oligos were further set to 0nt (crude end ligation), 1nt (TA ligation), 2nt, 3nt, 4nt, 5nt, and 6nt (that is, the oligo sequence in the original SEQ ID NO.5 was reduced from 6nt to 0nt in turn). The length of the complementary sequence in the adaptor was adjusted accordingly, and the ligation efficiency between the adapter and the adaptor containing oligos of different lengths was tested. The amount of T4 ligase (Vazyme#C301) added was 5 pmol, 18 pmol, and 360 pmol, respectively. The test results are shown in Figure 21.

[0755] The results show that, under different amounts of T4 ligase, the oligo length needs to reach at least 3nt to achieve a good ligation effect. When the amount of T4 ligase is 360pmol, oligos of 3-6nt can achieve a ligation efficiency of over 95%.

[0756] Example 6

[0757] Based on the Tn5-YU 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 Y-type linker forward insertion sequences containing different U bases are set as shown in the table below, and annealed with SEQ ID NO.7 to form Tn5-Y-U1, Tn5-Y-U2 and Tn5-Y-U3 complexes respectively.

[0758] Table 28

[0759] 10 ng gDNA was added and the library was constructed according to the procedure in Example 2. The library output is shown in Figure 22, and the peak shape of the library is shown in Figure 23.

[0760] As shown in Figure 22, replacing a single U base at different positions of the T base in the ME sequence has no effect on the library output, but replacing two U bases significantly reduces the output; as shown in Figure 23, replacing one or more U bases does not significantly change the library peak shape.

[0761] Example 7

[0762] Based on the library construction of the Tn5-oligo complex in Example 3 above, the number of U bases was further set to 2 and 3 to terminate the polymerization reaction. Specifically, SEQ ID NO.19 with 2 U bases was annealed with SEQ ID NO.8 to form the Tn5-oligo1 complex; and SEQ ID NO.20 with 3 U bases was annealed with SEQ ID NO.8 to form the Tn5-oligo2 complex. Correspondingly, the Tn5-oligo-linker used in library construction was also replaced with SEQ ID NO.21 and SEQ ID NO.22 with 2 terminal A bases, as follows:

[0763] Table 29

[0764] 10 ng gDNA was added for library construction, and the library was constructed according to the procedure in Example 3. The library output is shown in Figure 24, and the peak shape of the library is shown in Figure 25.

[0765] As shown in Figures 24 and 25, with the increase of U bases, the library output decreased significantly, the library was able to be broken normally, and the peak position did not change significantly.

[0766] Other sequences involved in this application are shown in the following table:

Claims

1. A linker oligonucleotide comprising a first strand and a second strand, wherein, The first strand, from its 5' end to its 3' end, includes an optional upstream sequencing immobilization sequence, an optional upstream sequencing primer sequence, and a transposase recognition core sequence. The second strand, from its 3' end to its 5' end, includes an optional reverse complementary sequence of a downstream sequencing immobilization sequence, an optional reverse complementary sequence of a downstream sequencing primer sequence, and an reverse complementary sequence of the transposase recognition core sequence. In this embodiment, 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 a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and no-base site, preferably a U base. The transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase, and optionally, it is an ME sequence, and optionally, as shown in SEQ ID NO.

23. Optionally, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, 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; more preferably, it is read1 or read2 from the Illumina platform; and even more preferably, 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; more preferably, it is read2 or read1 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 27 or 26. The linker oligonucleotide is a Y-type linker oligonucleotide, meaning that the inverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not inversely complementary when present, and the inverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not inversely complementary when present. Optionally, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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.

2. A linker oligonucleotide comprising a first strand and a second strand, wherein, The first strand contains a transposase recognition core sequence, and the second strand contains the reverse complementary sequence of the transposase recognition core sequence. In this embodiment, 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 a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and no-base site, preferably a U base. The transposase recognition core sequence is the transposase recognition core sequence of Tn5 transposase, and optionally, it is the ME sequence, and 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 contains 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 inverse complementary sequence of the optional downstream sequencing primer sequence and the inverse complementary sequence of the transposase recognition core sequence from the 3' end to the 5' end. In this embodiment, 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 a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and no-base site, preferably a U base. The transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase, and optionally, it is an ME sequence, and optionally, as shown in SEQ ID NO.

23. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence for the Illumina platform; more preferably, it is read1 or read2 from the Illumina platform; and even more preferably, 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; more preferably, it is read2 or read1 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 27 or 26. The linker oligonucleotide is a Y-type linker oligonucleotide, meaning that the inverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not inversely complementary when present. Optionally, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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.

4. A linker oligonucleotide comprising a first strand and a second strand, wherein, The first strand contains 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 inverse complementary sequence of the downstream sequencing primer sequence and the inverse complementary sequence of the transposase recognition core sequence from the 3' end to the 5' end. In this embodiment, 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 a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and no-base site, preferably a U base. The transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase, and optionally, it is an ME sequence, and optionally, as shown in SEQ ID NO.

23. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence for the Illumina platform; more preferably, it is read1 or read2 from the Illumina platform; and even more preferably, 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; more preferably, it is read2 or read1 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 27 or 26. The linker oligonucleotide is a Y-type linker oligonucleotide, meaning that the reverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary. Optionally, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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.

5. A linker oligonucleotide comprising a first strand and a second strand, wherein, The first strand contains 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. In this embodiment, 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 a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and no-base site, preferably a U base. The transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase, and optionally, it is an ME sequence, and optionally, as shown in SEQ ID NO.

23. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence for the Illumina platform; more preferably, it is read1 or read2 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 26 or 27. Optionally, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, the upstream tag sequence 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.

6. A linker oligonucleotide comprising a first strand and a second strand, wherein, The first strand, from its 5' end to its 3' end, comprises an upstream sequencing immobilization sequence, an upstream sequencing primer sequence, and a transposase recognition core sequence. The second strand, from its 3' end to its 5' end, comprises an inverse complementary sequence of an optional downstream sequencing immobilization sequence, an inverse complementary sequence of an optional downstream sequencing primer sequence, and an inverse complementary sequence of the transposase recognition core sequence. In this embodiment, 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 a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and no-base site, preferably a U base. The transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase, and optionally, it is an ME sequence, and optionally, as shown in SEQ ID NO.

23. Optionally, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, 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; more preferably, it is read1 or read2 from the Illumina platform; and even more preferably, 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; more preferably, it is read2 or read1 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 27 or 26. The linker oligonucleotide is a Y-type linker oligonucleotide, meaning that the inverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not inversely complementary when present, and the inverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not inversely complementary when present. Optionally, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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.

7. A linker oligonucleotide comprising a first strand and a second strand, wherein, The first strand, from its 5' end to its 3' end, contains an upstream sequencing immobilization sequence, an upstream sequencing primer sequence, and a transposase recognition core sequence. The second strand, from its 3' end to its 5' end, contains the reverse complementary sequence of the downstream sequencing immobilization sequence, the reverse complementary sequence of the downstream sequencing primer sequence, and the reverse complementary sequence of the transposase recognition core sequence. In this embodiment, 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 a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and no-base site, preferably a U base. The transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase, and optionally, it is an ME sequence, and optionally, as shown in SEQ ID NO.

23. Optionally, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, 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; more preferably, it is read1 or read2 from the Illumina platform; and even more preferably, 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; more preferably, it is read2 or read1 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 27 or 26. The linker oligonucleotide is a Y-type linker oligonucleotide, meaning that the inverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not inversely complementary, and the inverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not inversely complementary. Optionally, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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.

8. A linker 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 immobilization sequence, an upstream sequencing primer sequence, and a transposase recognition core sequence; the second strand contains the reverse complementary sequence of the transposase recognition core sequence. In this embodiment, 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 a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and no-base site, preferably a U base. The transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase, and optionally, it is an ME sequence, and optionally, as shown in SEQ ID NO.

23. Optionally, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence for the Illumina platform; more preferably, it is read1 or read2 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 26 or 27. Optionally, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, the upstream tag sequence 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.

9. An adaptor oligonucleotide comprising an optional first strand and a second strand, wherein, The first strand, from its 5' end to its 3' end, includes an optional upstream sequencing immobilization sequence, an upstream sequencing primer sequence, and an adaptor spacer sequence. The second strand, from its 3' end to its 5' end, includes an optional reverse complementary sequence of the downstream sequencing immobilization sequence, an optional reverse complementary sequence of the downstream sequencing primer sequence, an optional reverse complementary sequence of the adaptor spacer sequence, the 3' portion of the reverse complementary sequence of the transposase recognition core sequence, and an A base. Wherein, the 3' end portion of the inverse complementary sequence of the transposase recognition core sequence is inversely 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 immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, 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; more preferably, it is read1 or read2 from the Illumina platform; and even more preferably, 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; more preferably, it is read2 or read1 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 27 or 26. The adaptor oligonucleotide is a Y-type adaptor oligonucleotide, meaning that the inverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not inversely complementary when present, and the inverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not inversely complementary when present. Optionally, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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. Wherein, the length of the inverse complementary sequence of the adaptor spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

10. An adaptor oligonucleotide comprising a first strand and a second strand, wherein, The first strand, from its 5' end to its 3' end, includes an optional upstream sequencing immobilization sequence, an upstream sequencing primer sequence, and an adaptor spacer sequence. The second strand, from its 3' end to its 5' end, includes an optional reverse complementary sequence of the downstream sequencing immobilization sequence, a reverse complementary sequence of the downstream sequencing primer sequence, a reverse complementary sequence of the adaptor spacer sequence, the 3' portion of the reverse complementary sequence of the transposase recognition core sequence, and an A base. Wherein, the 3' end portion of the inverse complementary sequence of the transposase recognition core sequence is inversely complementary to the 5' end portion of the transposase recognition core sequence of the first strand of the linker 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 immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, 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; more preferably, it is read1 or read2 from the Illumina platform; and even more preferably, 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; more preferably, it is read2 or read1 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 27 or 26. The adaptor oligonucleotide is a Y-type adaptor oligonucleotide, meaning that the inverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not inversely complementary when present, and the inverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not inversely complementary when present. Optionally, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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. Wherein, the length of the inverse complementary sequence of the adaptor spacer sequence is 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 contains the upstream sequencing primer sequence and the adaptor 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 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 inverse complementary sequence of the transposase recognition core sequence is inversely complementary to the 5' end portion of the transposase recognition core sequence of the first strand of the linker 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 primer sequence is a sequencing primer sequence for the Illumina platform; more preferably, it is read1 or read2 from the Illumina platform; and even more preferably, 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; more preferably, it is read2 or read1 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 27 or 26. The adaptor oligonucleotide is a Y-type adaptor oligonucleotide, meaning that the reverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary. Optionally, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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. Wherein, the length of the inverse complementary sequence of the adaptor spacer sequence is 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, from its 5' end to its 3' end, comprises an upstream sequencing immobilization sequence, an upstream sequencing primer sequence, and an adaptor spacer sequence. The second strand, from its 3' end to its 5' end, comprises the reverse complementary sequence of the downstream sequencing immobilization sequence, the reverse complementary sequence of the downstream sequencing primer sequence, the reverse complementary sequence of the adaptor spacer sequence, the 3' portion of the reverse complementary sequence of the transposase recognition core sequence, and an A base. Wherein, the 3' end portion of the inverse complementary sequence of the transposase recognition core sequence is inversely complementary to the 5' end portion of the transposase recognition core sequence of the first strand of the linker 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 immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, 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; more preferably, it is read1 or read2 from the Illumina platform; and even more preferably, 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; more preferably, it is read2 or read1 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 27 or 26. The adaptor oligonucleotide is a Y-type adaptor oligonucleotide, meaning that the reverse complementary sequences 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 immobilization sequence and the downstream sequencing immobilization sequence are not reverse complementary. Optionally, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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. Wherein, the length of the inverse complementary sequence of the adaptor spacer sequence is 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, from the 3' end to the 5' end, contains the inverse complementary sequence of the downstream sequencing primer sequence, the 3' end portion of the inverse complementary sequence of the transposase recognition core sequence, and an A base. Wherein, the 3' end portion of the inverse complementary sequence of the transposase recognition core sequence is inversely complementary to the 5' end portion of the transposase recognition core sequence of the first strand of the linker 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 for the Illumina platform; more preferably, it is read2 or read1 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 27 or 26. The inverse complementary sequence of the downstream sequencing primer sequence is not inversely complementary to the upstream sequencing primer sequence of the linker oligonucleotide according to claim 5. Optionally, the second strand further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, the downstream tag sequence 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.

14. An adaptor oligonucleotide comprising a second strand, wherein, The second strand, from the 3' end to the 5' end, includes the inverse complementary sequence of the downstream sequencing primer sequence, the inverse complementary sequence of the downstream sequencing primer sequence, the 3' portion of the inverse complementary sequence of the transposase recognition core sequence, and an A base. Wherein, the 3' end portion of the inverse complementary sequence of the transposase recognition core sequence is inversely complementary to the 5' end portion of the transposase recognition core sequence of the first strand of the linker 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 immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 25 or 24. Optionally, the downstream sequencing primer sequence is a sequencing primer sequence for the Illumina platform; more preferably, it is read2 or read1 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 27 or 26. The inverse complementary sequence of the downstream sequencing primer sequence is not inversely complementary to the upstream sequencing primer sequence of the linker oligonucleotide according to claim 8, and the inverse complementary sequence of the downstream sequencing immobilization sequence is not inversely complementary to the upstream sequencing immobilization sequence of the linker oligonucleotide according to claim 8. Optionally, the second strand further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, the downstream tag sequence 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.

15. A method for constructing a sequencing library, comprising: (1) The target sequence is broken using a transposase complex containing the adapter oligonucleotide of claim 1; (2) The sequence is treated with a DNA polymerase, FEN1 enzyme, and ligase that are intolerant to base damage, optionally, the adapter oligonucleotide of claim 8 is added and denatured and annealed before ligase treatment; and (3) PCR is performed using an extension primer pair. The upstream primer of the extension primer pair contains, from the 5' end to the 3' end, an upstream sequencing immobilization sequence and the upstream sequencing primer sequence; the downstream primer of the extension primer pair contains, from the 5' end to the 3' end, a downstream sequencing immobilization sequence and the downstream sequencing primer sequence. Optionally, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 25 or 24. Optionally, the inverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not inversely complementary when they are present. Optionally, the upstream primer further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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' or 3' end of the downstream sequencing primer sequence, 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.

16. A method for constructing a sequencing library, comprising: (1) The target sequence is broken using a transposase complex containing the adapter oligonucleotide as described in claim 2; (2) The sequence is treated with a DNA polymerase, FEN1 enzyme, and ligase that are intolerant to base damage, wherein the adapter oligonucleotide as described in claim 11 is added and denatured and annealed before ligase treatment; and (3) PCR is performed using an extension primer pair. The upstream primer of the extension primer pair contains, from the 5' end to the 3' end, an upstream sequencing immobilization sequence and the upstream sequencing primer sequence; the downstream primer of the extension primer pair contains, from the 5' end to the 3' end, a downstream sequencing immobilization sequence and the downstream sequencing primer sequence. Optionally, the DNA polymerase intolerant to damaged bases is one or more of the following: U-base-intolerant DNA polymerases, including one or more of the following: 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 immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 25 or 24. Optionally, the inverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not inversely complementary when they are present. Optionally, the upstream primer further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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' or 3' end of the downstream sequencing primer sequence, 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.

17. A method for constructing a sequencing library, comprising: (1) The target sequence is broken using a transposase complex containing the adapter oligonucleotide according to claim 2; and (2) the sequence is treated with a DNA polymerase, FEN1 enzyme, and ligase that are intolerant to base damage, 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 following: U-base-intolerant DNA polymerases, including one or more of the following: 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) The target sequence was broken using a transposase complex containing the linker oligonucleotides described in claim 4; (2) the sequence was treated with DNA polymerase, FEN1 enzyme and ligase that are intolerant to base damage; and (3) PCR was performed using extension primer pairs. The upstream primer of the extension primer pair contains, from the 5' end to the 3' end, an upstream sequencing immobilization sequence and the upstream sequencing primer sequence; the downstream primer of the extension primer pair contains, from the 5' end to the 3' end, a downstream sequencing immobilization sequence and the downstream sequencing primer sequence. Optionally, the DNA polymerase intolerant to damaged bases is one or more of the following: U-base-intolerant DNA polymerases, including one or more of the following: 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 immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 25 or 24. Optionally, the inverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not inversely complementary when they are present. Optionally, the upstream primer further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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' or 3' end of the downstream sequencing primer sequence, 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.

19. A method for constructing a sequencing library, comprising: (1) The target sequence is broken using a transposase complex containing the adapter oligonucleotide as described in claim 5; (2) The sequence is treated with a DNA polymerase, FEN1 enzyme, and ligase that are intolerant to base damage, wherein the adapter oligonucleotide as described in claim 13 is added and denatured and annealed before ligase treatment; and (3) PCR is performed using an extension primer pair. The upstream primer of the extension primer pair contains, from the 5' end to the 3' end, an upstream sequencing immobilization sequence and the upstream sequencing primer sequence; the downstream primer of the extension primer pair contains, from the 5' end to the 3' end, a downstream sequencing immobilization sequence and the downstream sequencing primer sequence. Optionally, the DNA polymerase intolerant to damaged bases is one or more of the following: U-base-intolerant DNA polymerases, including one or more of the following: 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 immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 25 or 24. Optionally, the inverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not inversely complementary when they are present. Optionally, the upstream primer further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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' or 3' end of the downstream sequencing primer sequence, 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.

20. A method for constructing a sequencing library, comprising: (1) The target sequence was broken using a transposase complex containing the linker oligonucleotides according to claim 7; and (2) the sequence was treated with DNA polymerase, FEN1 enzyme and ligase that are intolerant to base damage. Optionally, the DNA polymerase intolerant to damaged bases is one or more of the following: U-base-intolerant DNA polymerases, including one or more of the following: 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) The target sequence is broken using a transposase complex containing the adapter oligonucleotide of claim 8; and (2) the sequence is treated with a DNA polymerase, FEN1 enzyme and ligase that are intolerant to base damage, wherein the adapter oligonucleotide of claim 14 is added and denatured and annealed before ligase treatment.

22. A reagent kit comprising... (1) The linker oligonucleotide according to claim 2, the adaptor oligonucleotide according to claim 11, and the extension primer pair; (2) The linker oligonucleotide according to claim 2 and the integrator oligonucleotide according to claim 12; (3) The linker oligonucleotide and extension primer pair according to claim 4; (4) The linker oligonucleotide according to claim 5, the integrator oligonucleotide according to claim 13, and the extension primer pair; (5) The linker oligonucleotide according to claim 7; or (6) The linker oligonucleotide according to claim 8 and the inductor oligonucleotide according to claim 14 in, The upstream primer of the extension primer pair contains, from the 5' end to the 3' end, an upstream sequencing immobilization sequence and the upstream sequencing primer sequence, and the downstream primer of the extension primer pair contains, from the 5' end to the 3' end, a downstream sequencing immobilization sequence and the downstream sequencing primer sequence. Optionally, the upstream primer further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, the upstream tag sequence being at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides in length, and / or the downstream primer further comprises a downstream tag sequence located at the 5' or 3' end of the downstream sequencing primer sequence, the downstream tag sequence being at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides in length. The kit also contains one or more of the following: (1) a transposase; (2) a DNA polymerase intolerant to base damage; (3) a FEN1 enzyme; and (4) a ligase. Optionally, the DNA polymerase intolerant to damaged bases is one or more of the following: U-base-intolerant DNA polymerases, including one or more of the following: 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. A linker oligonucleotide comprising a first strand and a second strand, wherein, The first strand, from its 5' end to its 3' end, includes an optional upstream sequencing immobilization 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, from its 3' end to its 5' end, includes an optional reverse complementary sequence of the downstream sequencing immobilization sequence, an optional reverse complementary sequence of the downstream sequencing primer sequence, an optional reverse complementary sequence of the linker spacer sequence, one or more optional A bases, and an optional reverse complementary sequence of the transposase recognition core sequence. The transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase, and more optionally, is an ME sequence, and more optionally, as shown in SEQ ID NO.

23. Optionally, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, 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; more preferably, it is read1 or read2 from the Illumina platform; and even more preferably, 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; more preferably, it is read2 or read1 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 27 or 26. Optionally, the linker oligonucleotide is a Y-type linker oligonucleotide, meaning that the reverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary when present, and the reverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not reverse complementary when present. Optionally, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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. Optionally, the damaged base is any one of a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and no-base site, preferably a U base. The length of the linker spacer sequence and its inverse complementary sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

24. A linker oligonucleotide comprising a first strand and a second strand, wherein, The first strand, from its 5' end to its 3' end, includes a linker spacer sequence, a damaged base, and a transposase recognition core sequence. The second strand, from its 3' end to its 5' end, includes, optionally, the inverse complementary sequence of the linker spacer sequence, an optional A base, and the inverse complementary sequence of the transposase recognition core sequence. The transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase, and more optionally, is an ME sequence, and more optionally, as shown in SEQ ID NO.

23. Optionally, the damaged base is any one of a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and no-base site, preferably a U base. The length of the linker spacer sequence and its inverse complementary sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

25. A linker oligonucleotide comprising a first strand and a second strand, wherein, The first strand, from its 5' end to its 3' end, includes a linker spacer sequence, a damaged base, and a transposase recognition core sequence. The second strand, from its 3' end to its 5' end, includes the inverse complementary sequence of the linker spacer sequence, an A base, and the inverse complementary sequence of the transposase recognition core sequence. The transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase, and optionally, is an ME sequence, or optionally, as shown in SEQ ID NO.

23. Optionally, the damaged base is any one of a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and no-base site, preferably a U base. The length of the linker spacer sequence and its inverse complementary sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

26. A linker oligonucleotide comprising a first strand and a second strand, wherein, The first strand contains a linker spacer sequence, damaged bases, and a transposase recognition core sequence from the 5' end to the 3' end. The second strand contains the inverse complementary sequence of the transposase recognition core sequence, wherein the transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase, and optionally, is an ME sequence, and optionally, as shown in SEQ ID NO.

23. Optionally, the damaged base is any one of a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and no-base site, preferably a U base. The length of the linker spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

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

23. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence for the Illumina platform; more preferably, it is read1 or read2 from the Illumina platform; and even more preferably, 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; more preferably, it is read2 or read1 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 27 or 26. Optionally, the linker oligonucleotide is a Y-type linker oligonucleotide, meaning that the reverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary when present. Optionally, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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. Optionally, the damaged base is any one of a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and no-base site, preferably a U base. The length of the linker spacer sequence and its inverse complementary sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

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

23. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence for the Illumina platform; more preferably, it is read1 or read2 from the Illumina platform; and even more preferably, 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; more preferably, it is read2 or read1 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 27 or 26. Optionally, the linker oligonucleotide is a Y-type linker oligonucleotide, meaning that the reverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary. Optionally, the damaged base is any one of a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and no-base site, preferably a U base. Optionally, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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. The length of the linker spacer sequence and its inverse complementary sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

29. A linker 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, damaged bases, and a transposase recognition core sequence. The second strand contains the inverse complementary sequence of the transposase recognition core sequence, wherein the transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase, and optionally, is an ME sequence, and optionally, as shown in SEQ ID NO.

23. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence for the Illumina platform; more preferably, it is read1 or read2 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 26 or 27. Optionally, the damaged base is any one of a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and no-base site, preferably a U base. Optionally, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, the upstream tag sequence 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. The length of the linker spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

30. A linker oligonucleotide comprising a first strand and a second strand, wherein, The first strand, from its 5' end to its 3' end, comprises an upstream sequencing immobilization sequence, an upstream sequencing primer sequence, a linker spacer sequence, a damaged base, and a transposase recognition core sequence. The second strand, from its 3' end to its 5' end, comprises an optional reverse complementary sequence of a downstream sequencing immobilization sequence, an optional reverse complementary sequence of a downstream sequencing primer sequence, an optional reverse complementary sequence of the linker spacer sequence, an optional A base, and an optional reverse complementary sequence of the transposase recognition core sequence. The transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase, and more optionally, is an ME sequence, and more optionally, as shown in SEQ ID NO.

23. Optionally, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, 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; more preferably, it is read1 or read2 from the Illumina platform; and even more preferably, 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; more preferably, it is read2 or read1 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 27 or 26. Optionally, the linker oligonucleotide is a Y-type linker oligonucleotide, meaning that the reverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary when present, and the reverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not reverse complementary when present. Optionally, the damaged base is any one of a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and no-base site, preferably a U base. Optionally, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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. The length of the linker spacer sequence and its inverse complementary sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

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

23. Optionally, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, 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; more preferably, it is read1 or read2 from the Illumina platform; and even more preferably, 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; more preferably, it is read2 or read1 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 27 or 26. Optionally, the linker oligonucleotide is a Y-type linker oligonucleotide, meaning that the inverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not inversely complementary, and the inverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not inversely complementary. Optionally, the damaged base is any one of a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and no-base site, preferably a U base. Optionally, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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. The length of the linker spacer sequence and its inverse complementary sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

32. A linker oligonucleotide comprising a first strand and a second strand, wherein, The first strand, from its 5' end to its 3' end, includes an upstream sequencing binding sequence, an upstream sequencing primer sequence, a linker spacer sequence, damaged bases, and a transposase recognition core sequence. The second strand includes the inverse complementary sequence of the transposase recognition core sequence, wherein the transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase, and optionally, is an ME sequence, and optionally, as shown in SEQ ID NO.

23. Optionally, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence for the Illumina platform; more preferably, it is read1 or read2 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 26 or 27. Optionally, the damaged base is any one of a U base, hypoxanthine, xanthine, methylated base, oxidized base, alkylated base, and no-base site, preferably a U base. Optionally, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, the upstream tag sequence 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. The length of the linker spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

33. An adaptor oligonucleotide comprising an optional first strand and a second strand, wherein, The first strand, from its 5' end to its 3' end, includes an optional upstream sequencing immobilization sequence, an upstream sequencing primer sequence, and an adapter spacer sequence. The second strand, from its 3' end to its 5' end, includes an optional reverse complementary sequence of a downstream sequencing immobilization sequence, an optional reverse complementary sequence of a downstream sequencing primer sequence, an optional reverse complementary sequence of the adapter spacer sequence, an optional reverse complementary sequence of the adapter spacer sequence, and an A base. Wherein, the inverse complementary sequence of the linker spacer sequence is inversely complementary to the linker spacer sequence of the first strand of the linker oligonucleotide according to claim 23. Optionally, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, 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; more preferably, it is read1 or read2 from the Illumina platform; and even more preferably, 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; more preferably, it is read2 or read1 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 27 or 26. The adaptor oligonucleotide is a Y-type adaptor oligonucleotide, meaning that the inverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not inversely complementary when present, and the inverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not inversely complementary when present. Optionally, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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. Wherein, the length of the inverse complementary sequence of the adaptor spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

34. An adaptor oligonucleotide comprising a first strand and a second strand, wherein, The first strand, from its 5' end to its 3' end, includes an optional upstream sequencing immobilization sequence, an upstream sequencing primer sequence, and an adaptor spacer sequence. The second strand, from its 3' end to its 5' end, includes an optional reverse complementary sequence of the downstream sequencing immobilization sequence, a reverse complementary sequence of the downstream sequencing primer sequence, a reverse complementary sequence of the adaptor spacer sequence, a reverse complementary sequence of the 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 immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, 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; more preferably, it is read1 or read2 from the Illumina platform; and even more preferably, 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; more preferably, it is read2 or read1 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 27 or 26. The adaptor oligonucleotide is a Y-type adaptor oligonucleotide, meaning that the inverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not inversely complementary when present, and the inverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not inversely complementary when present. Optionally, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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. Wherein, the length of the inverse complementary sequence of the adaptor spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

35. An adaptor oligonucleotide comprising a first strand and a second strand, wherein, The first strand contains, from the 5' end to the 3' end, the upstream sequencing primer sequence and the adaptor spacer sequence; the second strand contains, from the 3' end to the 5' end, the inverse complementary sequence of the downstream sequencing primer sequence, the inverse complementary sequence of the adaptor spacer sequence, the inverse complementary sequence of the adapter spacer sequence, and an A base. Wherein, the inverse complementary sequence of the linker spacer sequence is inversely 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 for the Illumina platform; more preferably, it is read1 or read2 from the Illumina platform; and even more preferably, 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; more preferably, it is read2 or read1 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 27 or 26. The adaptor oligonucleotide is a Y-type adaptor oligonucleotide, meaning that the reverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary. Optionally, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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. Wherein, the length of the inverse complementary sequence of the adaptor spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

36. An adaptor oligonucleotide comprising a first strand and a second strand, wherein, The first strand, from its 5' end to its 3' end, contains an upstream sequencing immobilization sequence, an upstream sequencing primer sequence, and an adapter spacer sequence. The second strand, from its 3' end to its 5' end, contains the reverse complementary sequence of the downstream sequencing immobilization sequence, 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 adapter spacer sequence, and an A base. Wherein, the inverse complementary sequence of the linker spacer sequence is inversely complementary to the linker spacer sequence of the first strand of the linker oligonucleotide according to claim 26. Optionally, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, 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; more preferably, it is read1 or read2 from the Illumina platform; and even more preferably, 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; more preferably, it is read2 or read1 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 27 or 26. The adaptor oligonucleotide is a Y-type adaptor oligonucleotide, meaning that the reverse complementary sequences 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 immobilization sequence and the downstream sequencing immobilization sequence are not reverse complementary. Optionally, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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. Wherein, the length of the inverse complementary sequence of the adaptor spacer sequence is 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 contains, from the 3' end to the 5' end, the inverse complementary sequence of the downstream sequencing primer sequence, the inverse complementary sequence of the adapter spacer sequence, and an A base. Wherein, the inverse complementary sequence of the linker spacer sequence is inversely complementary to the linker spacer sequence of the first strand of the linker oligonucleotide according to claim 29. Optionally, the downstream sequencing primer sequence is a sequencing primer sequence for the Illumina platform; more preferably, it is read2 or read1 from the Illumina platform; and even more preferably, 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 linker oligonucleotide according to claim 29. Optionally, the second strand further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, the downstream tag sequence 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.

38. An adaptor oligonucleotide comprising a second strand, wherein, The second strand, from the 3' end to the 5' end, contains the inverse complementary sequence of the downstream sequencing primer sequence, the inverse complementary sequence of the adapter spacer sequence, and an A base. Wherein, the inverse complementary sequence of the linker spacer sequence is inversely complementary to the linker spacer sequence of the first strand of the linker oligonucleotide according to claim 32. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 25 or 24. Optionally, the downstream sequencing primer sequence is a sequencing primer sequence for the Illumina platform; more preferably, it is read2 or read1 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 27 or 26. The inverse complementary sequence of the downstream sequencing primer sequence is not inversely complementary to the upstream sequencing primer sequence of the linker oligonucleotide according to claim 32, and the inverse complementary sequence of the downstream sequencing immobilization sequence is not inversely complementary to the upstream sequencing immobilization sequence of the linker oligonucleotide according to claim 32. Optionally, the second strand further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, the downstream tag sequence 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.

39. A method for constructing a sequencing library, comprising: (1) The target sequence was broken using a transposase complex containing the linker oligonucleotide as described in claim 23; (2) Treatment with DNA polymerase and ligase that are intolerant to base damage, optionally, adding the adaptor oligonucleotide according to claim 33 and denaturing and annealing before ligase treatment; and (3) optionally, performing PCR using extension primer pairs. The upstream primer of the extension primer pair contains, from the 5' end to the 3' end, an upstream sequencing immobilization sequence and the upstream sequencing primer sequence; the downstream primer of the extension primer pair contains, from the 5' end to the 3' end, a downstream sequencing immobilization sequence and the downstream sequencing primer sequence. Optionally, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 25 or 24. Optionally, the inverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not inversely complementary when they are present. Optionally, the DNA polymerase intolerant to damaged bases is one or more of the following: U-base-intolerant DNA polymerases, including one or more of the following: 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' or 3' end of the upstream sequencing primer sequence, 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' or 3' end of the downstream sequencing primer sequence, 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.

40. A method for constructing a sequencing library, comprising: (1) The target sequence is broken using a transposase complex containing the adapter oligonucleotide of claim 25; (2) The sequence is treated with a DNA polymerase, FEN1 enzyme, and ligase that are intolerant to base damage, wherein the adapter oligonucleotide of claim 35 is added and denatured and annealed before ligase treatment; and (3) PCR is performed using an extension primer pair. The upstream primer of the extension primer pair contains, from the 5' end to the 3' end, an upstream sequencing immobilization sequence and the upstream sequencing primer sequence; the downstream primer of the extension primer pair contains, from the 5' end to the 3' end, a downstream sequencing immobilization sequence and the downstream sequencing primer sequence. Optionally, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 25 or 24. Optionally, the inverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not inversely complementary when they are present. Optionally, the DNA polymerase intolerant to damaged bases is one or more of the following: U-base-intolerant DNA polymerases, including one or more of the following: 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' or 3' end of the upstream sequencing primer sequence, 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' or 3' end of the downstream sequencing primer sequence, 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.

41. A method for constructing a sequencing library, comprising: (1) The target sequence is broken using a transposase complex containing the adapter oligonucleotide of claim 25; and (2) the sequence is treated with a DNA polymerase, FEN1 enzyme, and ligase that are intolerant to base damage, wherein the adapter oligonucleotide of claim 36 is added and denatured and annealed prior to ligase treatment. Optionally, the DNA polymerase intolerant to damaged bases is one or more of the following: U-base-intolerant DNA polymerases, including one or more of the following: 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) The target sequence is broken using a transposase complex containing the adapter oligonucleotide of claim 26; (2) The sequence is treated with a DNA polymerase and ligase that are intolerant to base damage, wherein the adapter oligonucleotide of claim 35 is added before ligase treatment; and (3) PCR is performed using an extension primer pair. The upstream primer of the extension primer pair contains, from the 5' end to the 3' end, an upstream sequencing immobilization sequence and the upstream sequencing primer sequence; the downstream primer of the extension primer pair contains, from the 5' end to the 3' end, a downstream sequencing immobilization sequence and the downstream sequencing primer sequence. Optionally, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 25 or 24. Optionally, the inverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not inversely complementary when they are present. Optionally, the DNA polymerase intolerant to damaged bases is one or more of the following: U-base-intolerant DNA polymerases, including one or more of the following: 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' or 3' end of the upstream sequencing primer sequence, 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' or 3' end of the downstream sequencing primer sequence, 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.

43. A method for constructing a sequencing library, comprising: (1) The target sequence is broken using a transposase complex containing the adapter oligonucleotide of claim 26; and (2) the sequence is treated with a DNA polymerase and ligase that are intolerant to base damage, wherein the adapter oligonucleotide of claim 36 is added prior to the ligase treatment. Optionally, the DNA polymerase intolerant to damaged bases is one or more of the following: U-base-intolerant DNA polymerases, including one or more of the following: 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) The target sequence was broken using a transposase complex containing the linker oligonucleotides described in claim 28; (2) the sequence was treated with a DNA polymerase, FEN1 enzyme, and ligase that are intolerant to base damage; and (3) PCR was performed using an extension primer pair. The upstream primer of the extension primer pair contains, from the 5' end to the 3' end, an upstream sequencing immobilization sequence and the upstream sequencing primer sequence; the downstream primer of the extension primer pair contains, from the 5' end to the 3' end, a downstream sequencing immobilization sequence and the downstream sequencing primer sequence. Optionally, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 25 or 24. Optionally, the inverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not inversely complementary when they are present. Optionally, the upstream primer further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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' or 3' end of the downstream sequencing primer sequence, 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.

45. A method for constructing a sequencing library, comprising: (1) The target sequence was broken using a transposase complex containing the linker oligonucleotide of claim 29. (2) Treatment with DNA polymerase and ligase that are intolerant to base damage, wherein the adaptor oligonucleotide according to claim 37 is added before ligase treatment; and (3) PCR is performed using extension primer pairs. The upstream primer of the extension primer pair contains, from the 5' end to the 3' end, an upstream sequencing immobilization sequence and the upstream sequencing primer sequence; the downstream primer of the extension primer pair contains, from the 5' end to the 3' end, a downstream sequencing immobilization sequence and the downstream sequencing primer sequence. Optionally, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 25 or 24. Optionally, the inverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not inversely complementary when they are present. Optionally, the DNA polymerase intolerant to damaged bases is one or more of the following: U-base-intolerant DNA polymerases, including one or more of the following: 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' or 3' end of the upstream sequencing primer sequence, 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' or 3' end of the downstream sequencing primer sequence, 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.

46. ​​A method for constructing a sequencing library, comprising: (1) The target sequence was broken using a transposase complex containing the linker oligonucleotides according to claim 31; and (2) the sequence was treated with DNA polymerase, FEN1 enzyme and ligase that are intolerant to base damage. Optionally, the DNA polymerase intolerant to damaged bases is one or more of the following: U-base-intolerant DNA polymerases, including one or more of the following: 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) The target sequence is broken using a transposase complex containing the adapter oligonucleotide of claim 32; and (2) the sequence is treated with a DNA polymerase and ligase that are intolerant to base damage, wherein the adapter oligonucleotide of claim 38 is added prior to the ligase treatment. Optionally, the DNA polymerase intolerant to damaged bases is one or more of the following: U-base-intolerant DNA polymerases, including one or more of the following: 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 reagent kit comprising (1) The linker oligonucleotide according to claim 25, the integrator oligonucleotide according to claim 35, and the extension primer pair; (2) The linker oligonucleotide according to claim 25 and the integrator oligonucleotide according to claim 36; (3) The linker oligonucleotide according to claim 26, the adaptor oligonucleotide according to claim 35, and the extension primer pair; (4) The linker oligonucleotide according to claim 26 and the integrator oligonucleotide according to claim 36; (5) The linker oligonucleotide and extension primer pair according to claim 28; (6) The linker oligonucleotide according to claim 29, the adaptor oligonucleotide according to claim 37, and the extension primer pair; (7) The linker oligonucleotide according to claim 31; or (8) The linker oligonucleotide according to claim 32 and the inductor oligonucleotide according to claim 38 in, The upstream primer of the extension primer pair contains, from the 5' end to the 3' end, an upstream sequencing immobilization sequence and the upstream sequencing primer sequence, and the downstream primer of the extension primer pair contains, from the 5' end to the 3' end, a downstream sequencing immobilization sequence and the downstream sequencing primer sequence. Optionally, the upstream primer further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, the upstream tag sequence being at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides in length, and / or the downstream primer further comprises a downstream tag sequence located at the 5' or 3' end of the downstream sequencing primer sequence, the downstream tag sequence being at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides in length. The kit also contains one or more of the following: (1) a transposase; (2) a DNA polymerase intolerant to base damage; and (3) a ligase. Optionally, the DNA polymerase intolerant to damaged bases is one or more of the following: U-base-intolerant DNA polymerases, including one or more of the following: 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. A linker oligonucleotide comprising a first strand and a second strand, wherein, The first strand, from its 5' end to its 3' end, includes an optional upstream sequencing binding sequence, an optional upstream sequencing primer sequence, an optional adapter spacer sequence, one or more restriction sequences, and a transposase recognition core sequence. The second strand, from its 3' end to its 5' end, includes an optional reverse complementary sequence of the downstream sequencing binding sequence, an optional reverse complementary sequence of the downstream sequencing primer sequence, an optional reverse complementary sequence of the adapter spacer sequence, one or more restriction sequences, and an reverse complementary sequence of the transposase recognition core sequence. The restriction sequences, specifically restriction sequences one and two, constitute the recognition / cleavage site of the restriction endonuclease. The transposase recognition core sequence is the transposase recognition core sequence of the Tn5 transposase, and optionally, it is an ME sequence, and optionally, as shown in SEQ ID NO.

23. Optionally, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, 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; more preferably, it is read1 or read2 from the Illumina platform; and even more preferably, 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; more preferably, it is read2 or read1 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 27 or 26. Optionally, the linker oligonucleotide is a Y-type linker oligonucleotide, meaning that the reverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary when present, and the reverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not reverse complementary when present. Optionally, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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. The length of the linker spacer sequence and its inverse complementary sequence is 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 its 5' end to its 3' end, includes an optional upstream sequencing immobilization sequence, an upstream sequencing primer sequence, an optional adaptor spacer sequence, an optional adapter spacer sequence, and a restriction sequence three. The second strand, from its 3' end to its 5' end, includes an optional reverse complementary sequence of the downstream sequencing immobilization sequence, an optional reverse complementary sequence of the downstream sequencing primer sequence, an optional reverse complementary sequence of the adaptor spacer sequence, an optional reverse complementary sequence of the adapter spacer sequence, and a restriction sequence four. The joint spacer sequence is the same as that described in claim 49. Wherein, restriction sequence three and restriction sequence four constitute the post-cleavage sticky ends of the restriction endonuclease according to claim 49. Optionally, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, 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; more preferably, it is read1 or read2 from the Illumina platform; and even more preferably, 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; more preferably, it is read2 or read1 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 27 or 26. The adaptor oligonucleotide is a Y-type adaptor oligonucleotide, meaning that the inverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not inversely complementary when present, and the inverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not inversely complementary when present. Optionally, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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. Wherein, the length of the inverse complementary sequence of the adaptor spacer sequence is 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, from its 5' end to its 3' end, includes an upstream sequencing primer sequence, an optional adaptor spacer sequence, an optional adapter spacer sequence, and a restriction sequence three. The second strand, from its 3' end to its 5' end, includes the reverse complementary sequence of the downstream sequencing primer sequence, the reverse complementary sequence of the optional adaptor spacer sequence, the reverse complementary sequence of the optional adapter spacer sequence, and a restriction sequence four. The joint spacer sequence is the same as that described in claim 49. Wherein, restriction sequence three and restriction sequence four constitute the post-cleavage sticky ends of the restriction endonuclease according to claim 49. Optionally, the upstream sequencing primer sequence is a sequencing primer sequence for the Illumina platform; more preferably, it is read1 or read2 from the Illumina platform; and even more preferably, 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; more preferably, it is read2 or read1 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 27 or 26. The adaptor oligonucleotide is a Y-type adaptor oligonucleotide, meaning that the reverse complementary sequences of the upstream sequencing primer sequence and the downstream sequencing primer sequence are not reverse complementary. Optionally, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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. Wherein, the length of the inverse complementary sequence of the adaptor spacer sequence is 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 its 5' end to its 3' end, comprises an upstream sequencing immobilization sequence, an upstream sequencing primer sequence, an optional adaptor spacer sequence, an optional adapter spacer sequence, and a restriction sequence three. The second strand, from its 3' end to its 5' end, comprises the reverse complementary sequence of the downstream sequencing immobilization sequence, the reverse complementary sequence of the downstream sequencing primer sequence, the reverse complementary sequence of the optional adaptor spacer sequence, the reverse complementary sequence of the optional adapter spacer sequence, and a restriction sequence four. The joint spacer sequence is the same as that described in claim 49. Wherein, restriction sequence three and restriction sequence four constitute the post-cleavage sticky ends of the restriction endonuclease according to claim 49. Optionally, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, 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; more preferably, it is read1 or read2 from the Illumina platform; and even more preferably, 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; more preferably, it is read2 or read1 from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 27 or 26. The adaptor oligonucleotide is a Y-type adaptor oligonucleotide, meaning that the reverse complementary sequences 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 immobilization sequence and the downstream sequencing immobilization sequence are not reverse complementary. Optionally, the first chain further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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 second chain further comprises an inverse complementary sequence of a downstream tag sequence located at the 3' or 5' end of the inverse complementary sequence of the downstream sequencing primer sequence, 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. Wherein, the length of the inverse complementary sequence of the adaptor spacer sequence is at least 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides.

53. A method for constructing a sequencing library, comprising: (1) The target sequence was broken using a transposase complex containing the linker oligonucleotide of claim 49. (2) Treatment with DNA polymerase, the restriction endonuclease and ligase as described in claim 49, optionally, adding the adaptor oligonucleotide as described in claim 50 and denaturing and annealing before ligase treatment; and (3) optionally, performing PCR using extension primer pairs. The upstream primer of the extension primer pair contains, from the 5' end to the 3' end, an upstream sequencing immobilization sequence and the upstream sequencing primer sequence; the downstream primer of the extension primer pair contains, from the 5' end to the 3' end, a downstream sequencing immobilization sequence and the downstream sequencing primer sequence. Optionally, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 25 or 24. Optionally, the inverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not inversely complementary when they are present. Optionally, the upstream primer further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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' or 3' end of the downstream sequencing primer sequence, 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.

54. A method for constructing a sequencing library, comprising: (1) The target sequence was broken using a transposase complex containing the linker oligonucleotide of claim 49. (2) The sample is treated with DNA polymerase, the restriction endonuclease and ligase as described in claim 49, wherein the adaptor oligonucleotide as described in claim 51 is added before ligase treatment; and (3) PCR is performed using the extension primer pair. The upstream primer of the extension primer pair contains, from the 5' end to the 3' end, an upstream sequencing immobilization sequence and the upstream sequencing primer sequence; the downstream primer of the extension primer pair contains, from the 5' end to the 3' end, a downstream sequencing immobilization sequence and the downstream sequencing primer sequence. Optionally, the upstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P5 or P7 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 24 or 25. Optionally, the downstream sequencing immobilization sequence is a sequencing immobilization sequence from the Illumina platform; more preferably, it is a P7 or P5 sequence from the Illumina platform; and even more preferably, it is as shown in SEQ ID NO. 25 or 24. Optionally, the inverse complementary sequences of the upstream sequencing immobilization sequence and the downstream sequencing immobilization sequence are not inversely complementary when they are present. Optionally, the upstream primer further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, 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' or 3' end of the downstream sequencing primer sequence, 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.

55. A method for constructing a sequencing library, comprising: (1) The target sequence was broken using a transposase complex containing the linker oligonucleotide of claim 49. (2) The treatment is performed using DNA polymerase, the restriction endonuclease of claim 49, and the ligase, wherein the adaptor oligonucleotide of claim 52 is added before the ligase treatment.

56. A reagent kit comprising... (1) The linker oligonucleotide according to claim 49, the inductor oligonucleotide according to claim 51, and the extension primer pair; or (2) The linker oligonucleotide according to claim 49 and the inductor oligonucleotide according to claim 52 in, The upstream primer of the extension primer pair contains, from the 5' end to the 3' end, an upstream sequencing immobilization sequence and the upstream sequencing primer sequence, and the downstream primer of the extension primer pair contains, from the 5' end to the 3' end, a downstream sequencing immobilization sequence and the downstream sequencing primer sequence. Optionally, the upstream primer further comprises an upstream tag sequence located at the 5' or 3' end of the upstream sequencing primer sequence, the upstream tag sequence being at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides in length, and / or the downstream primer further comprises a downstream tag sequence located at the 5' or 3' end of the downstream sequencing primer sequence, the downstream tag sequence being at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides in length. The kit also contains one or more of the following: (1) a transposase; (2) a DNA polymerase; (3) the restriction endonuclease of claim 49; and (4) a ligase.

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