A method for constructing a transposase-based sequencing library with high sample utilization and related oligonucleotides

Abstract

The present invention provides a method for constructing a sequencing library based on a transposase with high sample utilization and related oligonucleotides, belonging to the field of biotechnology. The present invention redesigns a Y-shaped adapter and embeds it with a Tn5 transposase to obtain a transposition complex, thereby solving the problem of 50% fragment loss during Tn5 transposase library construction in the prior art, thereby improving the output rate of the library.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for improving sample utilization rate in construction of a second-generation sequencing library based on transposase and related oligonucleotides. Background Art

[0002] The second generation of gene sequencing has been widely used in various industries. A DNA molecule needs to be broken into appropriate sizes and added with sequencing adapters that match the sequencing matrix and sequencing primers before the subsequent second generation gene sequencing can be carried out. The process of breaking DNA molecules and adding adapters is called library construction. The conventional construction methods include: mechanical shearing, fragmentation enzyme shearing and Tn5 transposase shearing ( Figure 1 ). Among them, the Tn5 transposase method has the advantages of easy operation and short experimental process. However, since the current Tn5 transposase library construction method requires two Tn5 transposases to form a transposon complex with adapter1 and adapter2, this will lead to three possibilities for the embedded transposon, namely two homologous dimeric Tn5 transposons (two adapter1s or two adapter2s) and one heterologous Tn5 transposon (adapter1+adapter2) (Kia A, Gloeckner C, Osothprarop T, et al. Improved genome sequencing using an engineered transposase [J]. BMC Biotechnology, 2017, 17 (1): 6.; Li N, Jin K, Bai Y, et al. Tn5 Transposase Applied in Genomics Research [J]. International Journal of Molecular Sciences, 2020, 21 (21): 8329.).

[0003] After the three transposons above interrupt and cut the DNA, four possibilities will be generated according to the adapters added to the two ends of the DNA, namely, DNA 5' end: adapter1 or adapter2 and DNA 3' end: adapter1 or adapter2. This will result in the inability to perform PCR amplification when there are two adapter1s or adapter2s at both ends of the DNA due to the spontaneous complementary pairing at both ends, resulting in 50% of the Tn5 transposase fragmented DNA being unable to be successfully constructed and sequenced, and ultimately resulting in 50% of the library being lost (Kia A, Gloeckner C, Osothprarop T, et al. Improved genome sequencing using an engineered transposase [J]. BMC Biotechnology, 2017, 17 (1): 6.; Li N, Jin K, Bai Y, et al. Tn5 Transposase Applied in Genomics Research [J]. International Journal of Molecular Sciences, 2020, 21 (21): 8329.) Figure 2 ). SUMMARY OF THE INVENTION

[0005] Purpose of the Invention

[0006] 1. Solve the problem of 50% fragment loss in library construction using Tn5 transposase and improve the output rate of the library.

[0007] 2. Improve the embedding efficiency of Tn5 transposase and DNA adapter. Summary of the invention

[0008] 1. Redesign the DNA adapter so that the parts other than the Tn5 recognition core sequence (ME sequence) are unpaired, that is, the current Tn5 adapter is modified into a Y-type adapter.

[0009] 2. Perform stabilization modification on the ME sequence of the Y-type adapter, such as LNA (locked nucleic acid) modification.

[0010] 3. The constructed Y-type adapter and Tn5 enzyme are embedded, and finally only one Tn5 transposase complex is formed.

[0011] 4. Redesign the library construction process by using Bsu DNA polymerase or Klenow fragment without 3'→5' and 5'→3' exonuclease activity with T4 DNA ligase to prevent the linkers at the last two ends of the library from being strand displaced.

[0012] Beneficial Effects

[0013] 1. Use only one Y-type adapter (a pair of single-stranded oligonucleotides) to construct the Tn5 transposase complex. After DNA is interrupted, the gap is repaired and connected by DNA polymerase and ligase. Then PCR library amplification is performed. Since the adapters at both ends of the Y-type adapter are different, all DNA fragments can enter the library construction process, thus avoiding the problem of 50% fragment loss in the original Tn5 transposase library construction ( Figure 3 and Figure 4 ).

[0014] 2. Stabilize the ME sequence of the Y-type adapter, such as LNA (locked nucleic acid) modification, which greatly increases the annealing temperature of the adapter, making the Y-type adapter itself very stable and increasing the probability of Tn5 transposase embedding into a complex ( Figure 3 ).

[0015] 3. Since 50% of the fragments are not lost, the library output rate is increased, the required sequencing depth is reduced, and the uniformity is improved ( Figures 5 to 7 ), and the detection ability of rare targets, such as pathogenic microorganisms, is also greatly improved ( Figure 8 ). BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Schematic diagram of mechanical disruption, fragmentation enzyme disruption and Tn5 transposase disruption library construction technology;

[0017] Figure 2 : Schematic diagram of Tn5 transposase library construction with half of the library information lost;

[0018] Figure 3 :Schematic diagram of the construction principle of the Y-Tn5 transposon complex;

[0019] Figure 4 : Schematic diagram of the construction principle of Y-Tn5 transposon complex library;

[0020] Figure 5 :A: Comparison of library outputs constructed with different starting amounts using the Y-Tn5 complex and conventional Tn5; B: Comparison of library outputs constructed with different starting amounts using the Y-Tn5-2LNA complex and conventional Tn5; C: Comparison of library outputs constructed with different starting amounts using the Y-Tn5-1LNA complex and conventional Tn5;

[0021] Figure 6 : Distribution diagram of library lengths obtained by two library construction methods in Example 2;

[0022] Figure 7 : Comparison of library sequencing results obtained by the two library construction methods in Example 2;

[0023] Figure 8 : Example 3 Comparison of pathogen detection efficiency of two library construction methods. DETAILED DESCRIPTION OF THE INVENTION

[0024] In one aspect, the invention provides a pair of oligonucleotides comprising a first oligonucleotide and a second oligonucleotide, wherein:

[0025] The first oligonucleotide comprises a first adapter positive strand sequence and a Tn5-type transposon end sequence transfer strand sequence from the 5' end to the 3' end; and

[0026] The second oligonucleotide comprises a Tn5-type transposon end sequence non-transferred strand sequence and a second adapter negative strand sequence from the 5' end to the 3' end.

[0027] The first oligonucleotide is partially reverse complementary to the second oligonucleotide. For example, the Tn5 type transposon end sequence transfer strand sequence of the first oligonucleotide is completely reverse complementary to the Tn5 type transposon end sequence non-transfer strand sequence of the second oligonucleotide, and the first adapter positive strand sequence of the first oligonucleotide is not completely reverse complementary to the second adapter negative strand sequence of the second oligonucleotide or is not reverse complementary. In one embodiment, the first adapter positive strand sequence and / or the second adapter negative strand sequence have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides that are not complementary. In one embodiment, the first adapter positive strand sequence and / or the second adapter negative strand sequence have at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the nucleotides are not complementary. "Not completely reverse complementary or not reverse complementary" means that the first adapter positive strand sequence and the second adapter negative strand sequence do not hybridize to each other under stringent conditions.

[0028] In one embodiment, the first oligonucleotide and / or the second oligonucleotide may further comprise other elements related to sequencing.

[0029] In one embodiment, the first oligonucleotide comprises a first index sequence, such as between the first adaptor positive strand sequence and the Tn5-type transposon end sequence transferred strand sequence, and / or the second oligonucleotide comprises a second index sequence, such as between the Tn5-type transposon end sequence non-transferred strand sequence and the second adaptor negative strand sequence.

[0030] In one embodiment, the first oligonucleotide comprises a first sequencing primer anchor sequence, such as between the first adapter plus strand sequence and the Tn5-type transposon end sequence transferred strand sequence, and / or, the second oligonucleotide comprises a second sequencing primer target sequence, such as between the Tn5-type transposon end sequence non-transferred strand sequence and the second adapter minus strand sequence. In embodiments where the first oligonucleotide comprises the first index sequence, the first sequencing primer anchor sequence may be between the first index sequence and the Tn5-type transposon end sequence transferred strand sequence, or, preferably, between the first index sequence and the first adapter plus strand sequence. In embodiments where the second oligonucleotide comprises the second index sequence, the second sequencing primer target sequence may be between the second index sequence and the Tn5-type transposon end sequence non-transferred strand sequence, or, preferably, between the second index sequence and the second adapter minus strand sequence. In one embodiment, the first sequencing primer anchor sequence is reverse complementary to the first sequencing primer target sequence, and / or, the second sequencing primer anchor sequence is reverse complementary to the second sequencing primer target sequence. In one embodiment, a first sequencing primer having the first sequencing primer anchor sequence and / or a second sequencing primer having the second sequencing primer anchor sequence are added during the sequencing process to sequence the target sequence at the 5' end of the first sequencing primer target sequence and / or the second sequencing primer target sequence on the corresponding target template.

[0031] In one aspect, the invention provides a pair of oligonucleotides comprising a third oligonucleotide and a fourth oligonucleotide, wherein:

[0032] The third oligonucleotide comprises the first sequencing matrix-associated sequence positive strand sequence and the first adapter positive strand sequence from the 5' end to the 3' end; and

[0033] The fourth oligonucleotide comprises the second adaptor negative strand sequence and the second sequencing matrix-associated sequence negative strand sequence from the 5' end to the 3' end.

[0034] In one embodiment, the third oligonucleotide and / or the fourth oligonucleotide may further comprise other elements related to sequencing.

[0035] In one embodiment, the third oligonucleotide comprises a third index sequence, such as between the first sequencing matrix-associated sequence positive strand sequence and the first adapter positive strand sequence, and / or the fourth oligonucleotide comprises a fourth index sequence, such as between the second adapter negative strand sequence and the second sequencing matrix-associated sequence negative strand sequence.

[0036] In one embodiment, the third oligonucleotide comprises a third sequencing primer anchor sequence, for example, between the first sequencing matrix-related sequence positive strand sequence and the first adapter positive strand sequence, and / or, the fourth oligonucleotide comprises a fourth sequencing primer target sequence, for example, between the second adapter negative strand sequence and the second sequencing matrix-related sequence negative strand sequence. In embodiments where the third oligonucleotide comprises the third index sequence, the third sequencing primer anchor sequence may be between the third index sequence and the first adapter positive strand sequence, or, preferably, between the third index sequence and the first sequencing matrix-related sequence positive strand sequence. In embodiments where the fourth oligonucleotide comprises the fourth index sequence, the fourth sequencing primer target sequence may be between the fourth index sequence and the second adapter negative strand sequence, or, preferably, between the fourth index sequence and the second sequencing matrix-related sequence negative strand sequence. In one embodiment, the third sequencing primer anchor sequence is reverse complementary to the third sequencing primer target sequence, and / or, the fourth sequencing primer anchor sequence is reverse complementary to the fourth sequencing primer target sequence. In one embodiment, a third sequencing primer having the third sequencing primer anchor sequence and / or a fourth sequencing primer having the fourth sequencing primer anchor sequence is added during the sequencing process to sequence the target sequence at the 5' end of the third sequencing primer target sequence and / or the fourth sequencing primer target sequence on the corresponding target template.

[0037] In one aspect, the invention provides a pair of oligonucleotides comprising a first oligonucleotide and a second oligonucleotide, wherein:

[0038] The first oligonucleotide comprises a first sequencing matrix-associated sequence positive strand sequence and a Tn5-type transposon end sequence transfer strand sequence from the 5' end to the 3' end; and

[0039] The second oligonucleotide comprises a Tn5-type transposon end sequence non-transferred strand sequence and a second sequencing matrix-related sequence negative strand sequence from the 5' end to the 3' end.

[0040] The first oligonucleotide is partially reverse complementary to the second oligonucleotide. For example, the Tn5 type transposon end sequence transfer strand sequence of the first oligonucleotide is completely reverse complementary to the Tn5 type transposon end sequence non-transfer strand sequence of the second oligonucleotide, and the first sequencing matrix-related sequence positive strand sequence of the first oligonucleotide is not completely reverse complementary to the second sequencing matrix-related sequence negative strand sequence of the second oligonucleotide or is not reverse complementary. In one embodiment, the first sequencing matrix-related sequence positive strand sequence and / or the second sequencing matrix-related sequence negative strand sequence have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides that are not complementary. In one embodiment, the first sequencing matrix-associated sequence positive strand sequence and / or the second sequencing matrix-associated sequence negative strand sequence have at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the nucleotides are not complementary. "Not completely reverse complementary or not reverse complementary" means that the first sequencing matrix-associated sequence positive strand sequence and the second sequencing matrix-associated sequence negative strand sequence do not hybridize to each other under stringent conditions.

[0041] In one embodiment, the first oligonucleotide and / or the second oligonucleotide may further comprise other elements related to sequencing.

[0042] In one embodiment, the first oligonucleotide comprises a first index sequence, such as between a first sequencing matrix-associated sequence positive strand sequence and a Tn5-type transposon end sequence transferred strand sequence, and / or, the second oligonucleotide comprises a second index sequence, such as between a Tn5-type transposon end sequence non-transferred strand sequence and a second sequencing matrix-associated sequence negative strand sequence.

[0043] In one embodiment, the first oligonucleotide comprises a first sequencing primer anchor sequence, for example, between the first sequencing matrix-related sequence positive strand sequence and the Tn5-type transposon end sequence transferred strand sequence, and / or, the second oligonucleotide comprises a second sequencing primer target sequence, for example, between the Tn5-type transposon end sequence non-transferred strand sequence and the second sequencing matrix-related sequence negative strand sequence. In embodiments where the first oligonucleotide comprises the first index sequence, the first sequencing primer anchor sequence may be between the first index sequence and the Tn5-type transposon end sequence transferred strand sequence, or, preferably, between the first index sequence and the first sequencing matrix-related sequence positive strand sequence. In embodiments where the second oligonucleotide comprises the second index sequence, the second sequencing primer target sequence may be between the second index sequence and the Tn5-type transposon end sequence non-transferred strand sequence, or, preferably, between the second index sequence and the second sequencing matrix-related sequence negative strand sequence. In one embodiment, the first sequencing primer anchor sequence is reverse complementary to the first sequencing primer target sequence, and / or, the second sequencing primer anchor sequence is reverse complementary to the second sequencing primer target sequence. In one embodiment, a first sequencing primer having the first sequencing primer anchor sequence and / or a second sequencing primer having the second sequencing primer anchor sequence are added during the sequencing process to sequence the target sequence at the 5' end of the first sequencing primer target sequence and / or the second sequencing primer target sequence on the corresponding target template.

[0044] In one embodiment, the first oligonucleotide comprises a first adapter positive strand sequence, such as between a first sequencing substrate-associated sequence positive strand sequence and any one or more or even all of the aforementioned other elements, and / or the second oligonucleotide comprises a second adapter negative strand sequence, such as between a second sequencing substrate-associated sequence negative strand sequence and any one or more or even all of the aforementioned other elements.

[0045] In one embodiment, the first oligonucleotide comprises a third index sequence, such as between the first sequencing matrix-associated sequence positive strand sequence and the first adapter positive strand sequence, and / or the second oligonucleotide comprises a fourth index sequence, such as between the second adapter negative strand sequence and the second sequencing matrix-associated sequence negative strand sequence.

[0046] In one embodiment, the first oligonucleotide comprises a third sequencing primer anchor sequence, for example, between the first sequencing matrix-related sequence positive strand sequence and the first adapter positive strand sequence, and / or, the second oligonucleotide comprises a fourth sequencing primer target sequence, for example, between the second adapter negative strand sequence and the second sequencing matrix-related sequence negative strand sequence. In embodiments where the first oligonucleotide comprises the third index sequence, the third sequencing primer anchor sequence may be between the third index sequence and the first adapter positive strand sequence, or, preferably, between the third index sequence and the first sequencing matrix-related sequence positive strand sequence. In embodiments where the second oligonucleotide comprises the fourth index sequence, the fourth sequencing primer target sequence may be between the fourth index sequence and the second adapter negative strand sequence, or, preferably, between the fourth index sequence and the second sequencing matrix-related sequence negative strand sequence. In one embodiment, the third sequencing primer anchor sequence is reverse complementary to the third sequencing primer target sequence, and / or, the fourth sequencing primer anchor sequence is reverse complementary to the fourth sequencing primer target sequence. In one embodiment, a third sequencing primer having the third sequencing primer anchor sequence and / or a fourth sequencing primer having the fourth sequencing primer anchor sequence is added during the sequencing process to sequence the target sequence at the 5' end of the third sequencing primer target sequence and / or the fourth sequencing primer target sequence on the corresponding target template.

[0047] In one embodiment, the first oligonucleotide and / or the second oligonucleotide comprises a stabilizing modification.

[0048] In one embodiment, the stabilizing modification is a locked nucleic acid. In one embodiment, the first oligonucleotide and / or the second oligonucleotide each independently comprises one or more locked nucleic acid units, such as 1-100 locked nucleic acid units, and for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 25, 30, 35, 40, 45 or 50. In one embodiment, the locked nucleic acid is in the Tn5 type transposon end sequence transfer chain sequence and / or the Tn5 type transposon end sequence non-transfer chain sequence. In one embodiment, the first oligonucleotide and / or the second oligonucleotide are all (i.e. 100%) or part (e.g. 5%, 10%, 25%, 33%, 50%, 67%, 75%, 90%, 95% or 99%) locked nucleic acids. In one embodiment, the Tn5-type transposon end sequence transfer strand sequence and / or the Tn5-type transposon end sequence non-transfer strand sequence is entirely (i.e., 100%) or partially (e.g., 5%, 10%, 25%, 33%, 50%, 67%, 75%, 90%, 95% or 99%) locked nucleic acid. In one embodiment, the locked nucleic acid is distributed continuously or intermittently (e.g., n1 common nucleic acid units, followed by m1 locked nucleic acid units, followed by n2 common nucleic acid units, followed by m2 locked nucleic acid units, followed by n3 common nucleic acid units, followed by m3 locked nucleic acid units, and so on, each n is independently any natural number, and each m is independently any natural number, provided that the sum of all n and all m is less than the total length of the first oligonucleotide and / or the second oligonucleotide, or, less than the total length of the Tn5-type transposon end sequence transfer strand sequence and / or the Tn5-type transposon end sequence non-transfer strand sequence).In one embodiment, the Tn5-type transposon end sequence transferred strand sequence and / or the Tn5-type transposon end sequence non-transferred strand sequence has one of the following arrangements: L; LD; DL; LDL; LDLD; DLDL; LDLDL; LDLDLDLD; DLDLDLDL; LDLDLDLDL; LDLDLDLDLD; DLDLDLDLDL; LDLDLDLDLDLDL; LDLDLDLDLDLD; DLDLDLDLDLDL; LDLDLDLDLDLDLDL; LDLDLDLDLDLDLD; DLDLDLDLDLDL; LDLDLDLDLDLDLDLDL; LDLDLDLDLDLDLDLD; DLDLDLDLDLDLDL; LDLDLDLDLDLDLDLDL; LDLDLDLDLDLDLDLD; DLDLDLDLDLDLDL; LDLDLDLDLDLDLDLDLDL; LDLDLDLDLDLDLDLDLD; LDLDLDLDLDLDLDLD; DLDLDLDLDLDLDLDL; and LDLDLDLDLDLDLDLDLDLDLD, wherein L is a sequence of consecutive locked nucleic acids and D is a sequence of consecutive common nucleic acids. When used, each L is independently 1-19, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 sequences of consecutive locked nucleic acids; each D is independently 1-18, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 sequences of consecutive common nucleic acids. In one embodiment, the stabilizing modification is a modification of the internucleoside bond different from a conventional phosphate bond, such as a thiophosphate bond, a dithiophosphate bond, a phosphoramidate bond and / or a thiophosphoramidate bond. In one embodiment, the first oligonucleotide and / or the second oligonucleotide each independently comprises one or more internucleoside linkage modifications, such as 1-100 internucleoside linkage modifications, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 25, 30, 35, 40, 45 or 50.In one embodiment, the internucleoside bond modification is in the Tn5-type transposon end sequence transferred strand sequence and / or the Tn5-type transposon end sequence non-transferred strand sequence. In one embodiment, the first oligonucleotide and / or the second oligonucleotide are all (i.e. 100%) or part (e.g. 5%, 10%, 25%, 33%, 50%, 67%, 75%, 90%, 95% or 99%) internucleoside bond modifications. In one embodiment, the internucleoside bond modification is in the Tn5-type transposon end sequence transferred strand sequence and / or the Tn5-type transposon end sequence non-transferred strand sequence all (i.e. 100%) or part (e.g. 5%, 10%, 25%, 33%, 50%, 67%, 75%, 90%, 95% or 99%) internucleoside bond modification. In one embodiment, the internucleoside bond modifications are distributed continuously or intermittently (for example, n1 phosphate bonds, followed by m1 internucleoside bond modifications, followed by n2 phosphate bonds, followed by m2 internucleoside bond modifications, followed by n3 phosphate bonds, followed by m3 internucleoside bond modifications, and so on, each n is independently any natural number, and each m is independently any natural number, provided that the sum of all n and all m is less than the total length of the first oligonucleotide and / or the second oligonucleotide-1, or, less than the total length of the Tn5-type transposon end sequence transferred chain sequence and / or the Tn5-type transposon end sequence non-transferred chain sequence).

[0049] In one embodiment, the Tn5-type transposon end sequence transferred strand sequence and / or the Tn5-type transposon end sequence non-transferred strand sequence can be any Tn5-type transposon end sequence transferred strand sequence and / or Tn5-type transposon end sequence non-transferred strand sequence known in the art, for example, Tn5-type transposon end sequence transferred strand sequence and / or Tn5-type transposon end sequence non-transferred strand sequence used by current or future sequencing platforms (including but not limited to iontorrent platform, illumina platform and BGI platform). In one embodiment, the Tn5-type transposon end sequence transferred strand sequence is shown as AGATGTGTATAAGAGACAG (SEQ ID NO: 1), and / or, the Tn5-type transposon end sequence non-transferred strand sequence is shown as CTGTCTCTTATACACATCT (SEQ ID NO: 2).

[0050] In one embodiment, the first index sequence and / or the second index sequence and / or the third index sequence and / or the fourth index sequence can utilize any short oligonucleotide, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides in length. In one embodiment, the first index sequence and / or the second index sequence and / or the third index sequence and / or the fourth index sequence can utilize index sequences utilized by current or future sequencing platforms (including but not limited to ion torrent platform, illumina platform and BGI platform).

[0051] In one embodiment, the first sequencing primer and / or the second sequencing primer and / or the third sequencing primer and / or the fourth sequencing primer can utilize sequencing primers utilized by current or future sequencing platforms (including but not limited to iontorrent platform, illumina platform and BGI platform).

[0052] In one embodiment, the first adapter and / or the second adapter can utilize adapters utilized by current or future sequencing platforms (including but not limited to ion torrent platform, illumina platform and BGI platform). In one embodiment, the first adapter is different from the second adapter. In one embodiment, the first adapter is N5. In one embodiment, the first adapter positive strand sequence is as shown in GTCTCGTGGGCTCGG (SEQ ID NO: 3). In one embodiment, the second adapter is N7. In one embodiment, the second adapter negative strand sequence is as shown in TCGTCGGCAGCGTC (SEQ ID NO: 4). Vice versa.

[0053] In one embodiment, the first sequencing matrix-related sequence and / or the second sequencing matrix-related sequence can utilize the sequencing matrix-related sequence utilized by the current or future sequencing platform (including but not limited to ion torrent platform, illumina platform and BGI platform). In one embodiment, the first sequencing matrix-related sequence is different from the second sequencing matrix-related sequence.

[0054] In one embodiment, the first sequencing matrix-related sequence is P5 of the illumina platform. In one embodiment, the second sequencing matrix-related sequence is P7 of the illumina platform. Vice versa.

[0055] In one embodiment, the first sequencing substrate-related sequence is a P1 adapter of an ion torrent platform. In one embodiment, the second sequencing substrate-related sequence is an A adapter of an ion torrent platform. And vice versa.

[0056] In one embodiment, the first sequencing matrix-related sequence is an upstream splint sequence for single-end tag library construction of the MGI platform. In one embodiment, the second sequencing matrix-related sequence is a downstream splint sequence for single-end tag library construction of the MGI platform. Vice versa.

[0057] In one embodiment, the first sequencing matrix-related sequence is an upstream splint sequence for double-ended tag library construction on the MGI platform. In one embodiment, the second sequencing matrix-related sequence is a downstream splint sequence for double-ended tag library construction on the MGI platform. Vice versa.

[0058] In one embodiment, the nucleotide sequence of the first oligonucleotide is

[0059] GTCTCGTGGGCTCGGNNNNNNAGATGTGTATAAGAGACAG (SEQ ID NO: 5), and / or, the nucleotide sequence of the second oligonucleotide is as shown

[0060] CTGTCTCTTATACACATCTNNNNNNTCGTCGGCAGCGTC (SEQ ID NO: 6). In one embodiment, the first oligonucleotide is as shown in

[0061] (SEQ ID NO: 7), and / or, the second oligonucleotide is as shown in

[0062] (SEQ ID NO: 8). In one embodiment, the first oligonucleotide is

[0063] (SEQ ID NO: 9), and / or, the second oligonucleotide is as shown in

[0064] (SEQ ID NO: 10). In one embodiment, the first oligonucleotide is

[0065] (SEQ ID NO: 11), and / or, the second oligonucleotide is as shown in

[0066] (SEQ ID NO: 12). The bold bases are ME sequences, the underlined bases are LNA modified bases, the italic bases are N5 or N7 sequences, and NNNNNN is an index sequence (N is any nucleotide, i.e., A, T, G or C).

[0067] In one embodiment, the first oligonucleotide and / or the second oligonucleotide are single stranded.In one embodiment, the 3' end of the first oligonucleotide is connected to the 5' end of the second oligonucleotide, optionally via a cleavable linker sequence, such as a restriction enzyme recognition site.

[0068] In one aspect, the present invention provides a complex comprising a Tn5 transposase and a pair of oligonucleotides of the present invention. In one embodiment, the Tn5 transposase is any Tn5 transposase known in the art or its active mutant. In one embodiment, the Tn5 transposase is a highly active Tn5 transposase (see, for example, WO2001 / 009363, US 5,925,545, US 5,965,443, US 7,083,980 and US7,608,434, and Goryshin and Reznikoff, J.Biol.Chem.273:7367,1998) or its active mutant. In one embodiment, the Tn5 transposase is the Tn5 transposase described in WO2015 / 160895. In one embodiment, the Tn5 transposase is a fusion protein. In one embodiment, the Tn5 transposase fusion protein comprises a fused elongation factor Ts (Tsf) tag. In one embodiment, the Tn5 transposase is a hyperactive Tn5 transposase comprising mutations at amino acids 54, 56, and 372 relative to the wild-type sequence.

[0069] In one aspect, the present invention provides a method for constructing a sequencing library, which comprises treating a target DNA with the complex of the present invention. In one embodiment, the method of the present invention comprises: (1) treating the target DNA with the complex of the present invention to fragment it and label it; (2) treating the set of labeled target DNA fragments obtained in step (1) with a DNA polymerase and a DNA ligase without 5'→3' and / or 3'→5' exonuclease activity to repair it; and (3) optionally, amplifying the repaired set of labeled target DNA fragments by PCR. In one embodiment, step (1) is incubated at 40-70°C, such as 45-65°C, such as 50-60°C, such as 55°C for 1-25 minutes, such as 3-20 minutes, such as 5-15 minutes, such as 10 minutes. In one embodiment, step (2) is incubation at 37°C for 10-50 minutes, such as 20-40 minutes, such as 30 minutes, followed by incubation at 16°C for 10-50 minutes, such as 20-40 minutes, such as 30 minutes, and then incubation at 72°C for 10-30 minutes, such as 15-25 minutes, such as 20 minutes, such as incubation at 37°C for 30 minutes, followed by incubation at 16°C for 30 minutes, and then incubation at 72°C for 20 minutes. In one embodiment, the method of the present invention further comprises recovering the labeled target DNA fragment set repair product obtained in step (2) after step (2). In one embodiment, the method of the present invention further comprises recovering / purifying the labeled target DNA fragment set repair / amplification product obtained in step (3) after step (3). In one embodiment, the DNA polymerase without 3'→5' exonuclease activity is Klenow fragment. In one embodiment, the DNA ligase is T4 DNA ligase.

[0070] Best Mode (Example)

[0071] Embodiment 1:

[0072] Preparation of Y-adapter-Tn5 transposase complex

[0073] Oligo A and Oligo B with locked nucleic acid modification were synthesized at Sangon Biotech (Shanghai). Oligo A and Oligo B were dissolved in PBS to 10 μM respectively, and 10 μl Oligo A + 10 μl Oligo B were taken to form reaction 1. Reaction 1 was vortexed and thoroughly mixed, and centrifuged briefly to return the solution to the bottom of the tube, and placed in a PCR instrument to perform the following reaction procedure:

[0074] Heat cover 105℃ 75℃ 15min 60℃ 10min 50℃ 10min 40℃ 10min 25℃ 30min

[0075] After the reaction was completed, the product of reaction 1 was named Tn5YAdapter.

[0076] Then add the following reaction components in the sterilized PCR tube in sequence:

[0077] Components 2μg system Tn5 (500 ng / μl)* 4μl Tn5Y Adapter 7μl PBS 39μl

[0078] *Tn5 was expressed in-house from the psfTn5 plasmid (Addgene plasmid #79107).

[0079] Use a pipette to gently pipette 20 times to mix thoroughly, and place at 30°C to react for 1 hour. The reaction product is named Y-Tn5 complex and stored at -30 to -15°C.

[0080] The Oligo A sequence is SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11, and the Oligo B sequence is SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12. SEQ ID NO: 7 and SEQ ID NO: 8 are used to form a Y-Tn5 complex, SEQ ID NO: 9 and SEQ ID NO: 10 are used to form a Y-Tn5-2LNA complex, and SEQ ID NO: 11 and SEQ ID NO: 12 are used to form a Y-Tn5-1LNA complex.

[0081] Embodiment 2:

[0082] In this example, 293 cell gDNA genome was used as an exemplary template, and the "conventional Tn5 product library construction" and "Y-Tn5 complex library construction" methods were used for experimental comparison to illustrate the method steps and beneficial effects of the present invention. In this example, 100 pg, 1 ng, and 10 ng of purified gDNA were taken as the starting DNA template, respectively. The experimental results are shown in Figure 5-Figure 7 ,in Figure 5 Panel A shows the experimental results of the Y-Tn5 complex, Panel B shows the experimental results of the Y-Tn5-2LNA complex, and Panel C shows the experimental results of the Y-Tn5-1LNA complex. The specific operations are as follows:

[0083] 1. Conventional Tn5 product library construction (using Novozyme product TD502)

[0084] DNA fragmentation

[0085] Thaw 5×TTBL at room temperature, mix by inverting and set aside. Check whether 5×TS is at room temperature and tap the tube wall to check for precipitation. If there is precipitation, heat at 37℃ and vortex to mix, and the precipitation will dissolve.

[0086] Prepare the following reaction system in a sterilized PCR tube:

[0087] Components volume 5×TTBL 4μl gDNA Xμl TTE Mix V5 5μl <![CDATA[ddH2O]]> To 20 μl

[0088] Mix thoroughly by pipetting up and down 20 times.

[0089] Place the reaction tube in the PCR instrument and run the following reaction program:

[0090] Heat cover 105℃ 55℃ 10min 10℃ Hold

[0091] PCR enrichment

[0092] After the reaction is completed, add 5 μl 5×TS to the product immediately, use a pipette to gently blow and mix thoroughly, place at room temperature for 5 minutes, then immediately prepare the PCR system and perform the PCR reaction. The specific system and reaction are as follows:

[0093] Components volume <![CDATA[ddH2O]]> 4μl Fragmentation products 25μl 5×TAB 10μl N5XX* 5μl N7XX* 5μl TAE 1μl

[0094] *8 types of N5XX and 12 types of N7XX are from Nanjing Novozymes product TD202.

[0095] After mixing the above system by pipetting, the following reaction is carried out:

[0096]

[0097] PCR product purification

[0098] Vortex and mix the DNA purification magnetic beads (N411, a product of Nanjing Novozymes) and pipette 25 μl of the magnetic beads into the above PCR reaction product. Vortex or pipette 10 times to ensure that the entire system is uniform, and incubate at room temperature for 5 minutes.

[0099] Centrifuge the reaction tube briefly and place it on a magnetic stand to separate the magnetic beads and liquid. After the solution becomes clear (about 5 minutes), carefully transfer the supernatant to a new centrifuge tube, taking care not to disturb the magnetic beads.

[0100] Pipette 7.5 μl of magnetic beads into the supernatant, vortex or pipette 10 times to ensure the uniformity of the whole system, incubate at room temperature for 5 minutes, place on a magnetic stand until the solution is clear, and remove the supernatant.

[0101] Keep the centrifuge tube on the magnetic stand, add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant. Repeat this step for a total of two rinses.

[0102] Keep the centrifuge tube on the magnetic rack at all times and air dry it with the lid open for 3-5 minutes.

[0103] After the magnetic beads are dried, remove the centrifuge tube from the magnetic rack, add 22 μl of sterile ultrapure water for elution, vortex or use a pipette to blow 10 times to fully mix the magnetic beads, and incubate at room temperature for 5 minutes.

[0104] Briefly centrifuge the PCR tube and place it on a magnetic rack to separate the magnetic beads and liquid. After the solution becomes clear (about 5 minutes), carefully transfer 20 μl of the supernatant to a new EP tube and store it at -30 to -15°C.

[0105] Library quality detection

[0106] The prepared library was tested for library length distribution on Agilent 2100 Bioanalyzer.

[0107] Next-generation sequencing

[0108] The library was diluted to 5 ng / μl, and 15 μl was sent to Nanjing Shihe Gene Biotechnology Co., Ltd. for sequencing using the HiseqX sequencing instrument.

[0109] 2. Y-Tn5 complex library construction (using some reagents in Novozyme product TD502)

[0110] DNA fragmentation

[0111] Thaw 5×TTBL at room temperature, mix by inverting and set aside. Check whether 5×TS is at room temperature and tap the tube wall to check for precipitation. If there is precipitation, heat at 37℃ and vortex to mix, and the precipitation will dissolve.

[0112] Prepare the following reaction system in a sterilized PCR tube:

[0113] Components volume 5×TTBL 4μl gDNA Xμl Y-Tn5 complex 1μl <![CDATA[ddH2O]]> To 20μl

[0114] Mix thoroughly by pipetting up and down 20 times.

[0115] Place the reaction tube in the PCR instrument and run the following reaction program:

[0116] Heat cover 105℃ 55℃ 10min 10℃ Hold

[0117] DNA fragment repair ligation

[0118] Immediately after the reaction is completed, add 5 μl 5×TS to the product, use a pipette to gently blow and mix thoroughly, place at room temperature for 5 minutes, and then immediately prepare the repair connection system and react. The specific system and reaction are as follows:

[0119]

[0120] NEBuffer 2: 50mM NaCl, 10mM Tris-HCl, 10mM MgCl2, 1mM DTT, pH7.9@25℃.

[0121] After mixing by pipetting, proceed with the following reaction:

[0122] temperature time 105℃ Heat cover 37℃ 30min 16℃ 30min 72℃ 20min 4℃ Hold

[0123] Repair ligation product purification

[0124] Vortex the DNA purification magnetic beads (N411, a product of Nanjing Novozymes) and pipette 60 μl into the above-mentioned repair ligation product. Vortex or pipette 10 times to ensure that the entire system is uniform, and incubate at room temperature for 5 minutes.

[0125] Centrifuge the reaction tube briefly and place it on a magnetic stand to separate the magnetic beads and liquid. After the solution becomes clear (about 5 minutes), carefully remove the supernatant, taking care not to disturb the magnetic beads.

[0126] Keep the centrifuge tube on the magnetic stand, add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant. Repeat this step for a total of two rinses.

[0127] Keep the centrifuge tube on the magnetic rack at all times and air dry it with the lid open for 3-5 minutes.

[0128] After the magnetic beads are dried, remove the centrifuge tube from the magnetic rack, add 26 μl of sterile ultrapure water for elution, vortex or use a pipette to mix the magnetic beads 10 times, and incubate at room temperature for 5 minutes.

[0129] Briefly centrifuge the PCR tube and place it on a magnetic rack to separate the magnetic beads and liquid. After the solution becomes clear (about 5 minutes), carefully transfer 25 μl of the supernatant to a new EP tube and store it at -30 to -15°C.

[0130] PCR enrichment

[0131] The library construction method is the same as the conventional Tn5 product method mentioned above.

[0132] PCR product purification

[0133] The library construction method is the same as the conventional Tn5 product method mentioned above.

[0134] Library quality detection

[0135] The library construction method is the same as the conventional Tn5 product method mentioned above.

[0136] Next-generation sequencing

[0137] The library construction method is the same as the conventional Tn5 product method mentioned above.

[0138] Experimental results

[0139] Figure 5: Panel A: The yield of the Y-Tn5 complex library is much better than that of the library prepared with conventional Tn5 when the input amount is different. The output of the library with an input amount of 100 pg differs by about 10 times; Panel B: A comparison of the output of libraries constructed with different starting amounts using the Y-Tn5-2LNA complex and conventional Tn5; Panel C: A comparison of the output of libraries constructed with different starting amounts using the Y-Tn5-1LNA complex and conventional Tn5.

[0140] Figure 6 : The library distribution shows that the fragment size distribution of Y-Tn5 is consistent with that of conventional Tn5 libraries, and both can effectively interrupt the genome.

[0141] Figure 7 : Sequencing analysis results show that Y-Tn5 has doubled the library uniformity compared to conventional Tn5 (the smaller the Uniformity value, the better), and significantly reduced the requirement for sequencing depth (the number of reads that require a depth of 10X or more to detect accounts for about 20%, while the ordinary TN5 is about 95%), while there is no difference in conventional mapping rate, coverage, and abnormal alignment (softclip) indicators.

[0142] Embodiment 3:

[0143] In this example, the efficiency of pathogen detection was evaluated by two methods: "conventional Tn5 product library construction" and "Y-Tn5 complex library construction". The templates used in the example were 293 cell gDNA with different types of pathogens added, namely (100 pg 293 gDNA + 0.1% λDNA), (1 ng 293 gDNA + 0.1% λDNA), (10 ng 293 gDNA + 0.1% λDNA), (100 pg 293 gDNA + 0.1% Staphylococcus aureus DNA), (1 ng 293 gDNA + 0.1% Staphylococcus aureus DNA), (10 ng 293 gDNA + 0.1% Staphylococcus aureus DNA), (100 pg 293 gDNA + 0.1% halophilic archaea DNA), (1 ng 293 gDNA + 0.1% halophilic archaea DNA), (10 ng 293gDNA+0.1% halophilic archaea DNA), the experimental method and steps are the same as in Example 2, and the experimental results are shown in Figure 8 .

[0144] Experimental results

[0145] Figure 8In terms of pathogen detection ability, the detection ability of Y-Tn5 for λDNA, Staphylococcus aureus DNA, and halophilic archaea DNA is better than that of conventional Tn5 for pathogen detection. Specifically, under the same amount of data, the number of pathogen DNA reads in the Y-Tn5 group is higher, and the ratio to the host is closer to the actual input ratio (0.1%).

Claims

1. An oligonucleotide pair comprising a first oligonucleotide and a second oligonucleotide, wherein: The first oligonucleotide comprises a first adapter positive strand sequence and a Tn5-type transposon end sequence transfer strand sequence from the 5' end to the 3' end; and The second oligonucleotide comprises a Tn5-type transposon end sequence non-transferred strand sequence and a second adapter negative strand sequence from the 5' end to the 3' end, The Tn5-type transposon end sequence transferred strand sequence and the Tn5-type transposon end sequence non-transferred strand sequence comprise a stabilizing modification, wherein the stabilizing modification is a locked nucleic acid, wherein the Tn5-type transposon end sequence transferred strand sequence and the Tn5-type transposon end sequence non-transferred strand sequence have a layout of LDLDL, wherein L is a sequence of a locked nucleic acid, D is a sequence of a continuous common nucleic acid, wherein each L is independently 1 locked nucleic acid, The first adaptor is different from the second adaptor, The first oligonucleotide and the second oligonucleotide are single-stranded.

2. The oligonucleotide pair of claim 1, wherein: The first oligonucleotide comprises a first index sequence between the first adaptor positive strand sequence and the Tn5-type transposon end sequence transferred strand sequence, and / or the second oligonucleotide comprises a second index sequence between the Tn5-type transposon end sequence non-transferred strand sequence and the second adaptor negative strand sequence.

3. The oligonucleotide pair of claim 1, wherein: The first oligonucleotide comprises a first sequencing primer anchor sequence between the first adaptor positive strand sequence and the Tn5-type transposon end sequence transferred strand sequence, and / or the second oligonucleotide comprises a second sequencing primer target sequence between the Tn5-type transposon end sequence non-transferred strand sequence and the second adaptor negative strand sequence.

4. The oligonucleotide pair of claim 2, wherein: The first oligonucleotide comprises a first sequencing primer anchor sequence between the first adapter positive strand sequence and the first index sequence, and / or the second oligonucleotide comprises a second sequencing primer target sequence between the second index sequence and the second adapter negative strand sequence. 5 . A Tn5 transposase complex, formed by Tn5 transposase and the oligonucleotide pair according to any one of claims 1 to 4 . 6 . A kit comprising the oligonucleotide pair according to any one of claims 1 to 4 or the Tn5 transposase complex according to claim 5 .

7. The kit of claim 6, further comprising a second oligonucleotide pair, the second oligonucleotide pair comprising a third oligonucleotide and a fourth oligonucleotide, wherein: The third oligonucleotide comprises a first sequencing matrix-associated sequence positive strand sequence and the first adapter positive strand sequence from the 5' end to the 3' end; and The fourth oligonucleotide comprises the second adaptor negative strand sequence and the second sequencing matrix-associated sequence negative strand sequence from the 5′ end to the 3′ end.

8. The kit of claim 7, wherein: The third oligonucleotide comprises a third index sequence between the first sequencing matrix-associated sequence positive strand sequence and the first adapter positive strand sequence, and / or the fourth oligonucleotide comprises a fourth index sequence between the second adapter negative strand sequence and the second sequencing matrix-associated sequence negative strand sequence.

9. The kit of claim 7, wherein: The third oligonucleotide comprises a third sequencing primer anchor sequence between the first sequencing matrix-associated sequence positive strand sequence and the first adapter positive strand sequence, and / or the fourth oligonucleotide comprises a fourth sequencing primer target sequence between the second adapter negative strand sequence and the second sequencing matrix-associated sequence negative strand sequence.

10. The kit of claim 8, wherein: The third oligonucleotide comprises a third sequencing primer anchor sequence between the first sequencing matrix-associated sequence positive strand sequence and the third index sequence, and / or the fourth oligonucleotide comprises a fourth sequencing primer target sequence between the fourth index sequence and the second sequencing matrix-associated sequence negative strand sequence.

11. A method for constructing a sequencing library, comprising the following steps: (1) contacting a sample containing double-stranded DNA with the Tn5 transposase complex of claim 5; (2) contacting the product of step (1) with a DNA polymerase and a DNA ligase without exonuclease activity; and (3) Optionally, amplifying the product of step (2) using a second oligonucleotide pair in the kit of any one of claims 7 to 10.

12. The method of claim 11, wherein: The DNA polymerase without exonuclease activity is Bsu DNA polymerase or Klenow fragment, and / or, the DNA ligase is T4 DNA ligase.

Images