WO2014089797A1 - Locked nucleic acid-modified dna fragment for high-throughput sequencing - Google Patents

Abstract

Disclosed is a locked nucleic acid-modified DNA fragment for high-throughput sequencing. The DNA fragment is selected from one, two or three of a linker, a PCR primer and a sequencing primer, and the linker, PCR primer and sequencing primer in the DNA fragment contain locked nucleic acids. Also disclosed are a high-throughput sequencing method, a DNA library construction method and a DNA library sequencing method, comprising a step of performing locked nucleic acid modification on a linker, a PCR primer and/or a sequencing primer. Also disclosed is the use of the DNA fragment in high-throughput sequencing, DNA library construction or DNA library sequencing.

Description

 Locked nucleic acid modified DNA fragment for high throughput sequencing

 The present invention relates to high throughput sequencing related techniques, and in particular to LNA modified DNA fragments for high throughput sequencing, including DNA linkers, PCR primers and/or sequencing primers. The present invention also relates to a high throughput sequencing method comprising a DNA library preparation and sequencing method for high throughput sequencing, the DNA library preparation and sequencing method comprising the step of using an LNA modified DNA fragment. Background technique

Locked nucleic acid (LNA) is a synthetic antisense oligonucleotide, a special bicyclic nucleotide derivative in which the nucleotide residue of the ribose ring (β-D-) The 2,-oxygen and 4,-carbon of ribofuranose form a fluorenylene linkage by shrinkage, and the structure contains one or more 2'-0,4'-C-fluorenylene-β-D-ribofuranosyl The monomer, the 2'-0 position and the 4'-C position of ribose form an oxysulfinyl bridge, a sulfinylene bridge or an amine fluorene bridge through different shrinkage, and are connected in a ring shape, and the ring bridge is locked. The N-configuration of the furanose C3'-endotype reduces the flexibility of the ribose structure and increases the stability of the local structure of the phosphate backbone. Because LNA and DNA/RNA have the same phosphate skeleton in structure, they have good recognition ability and strong affinity for DNA and RNA (Li Shengmao, Xu Xiang, Liang Huaping, Research progress in locked nucleic acid, Physiological science) Progress, 2003, 34 ( 4 ), 319-323 ) ₀

 With the application and development of high-throughput sequencing technology, DNA sequencing technology is also becoming more mature. The commonly used DNA library construction technology is prone to the generation of linkers and primer dimers, which affects the quality of the database and increases the amount of invalid data. At present, there are few reports on the direct use of LNA technology for high-throughput sequencing technology. Reduction of non-insert sequence reads by dimer eliminator LNA oligonucleotide for small RNA deep sequencing, BioTechniques, 49:751-755 (October 2010) doi 10.2144/000113516) The LNA probe was used to remove self-ligated fragments from the small RNA library to reduce the proportion of non-insert data in the library. Therefore, the ability to combine LNA technology with DNA high-throughput sequencing technology to improve library building efficiency and increase sequencing accuracy is an urgent problem in this field. Summary of the invention

 Through intensive efforts, the inventors have surprisingly found that high-throughput sequencing can be greatly improved if the nucleic acid (LNA) modification of the linker, PCR primers and/or sequencing primers involved in high throughput sequencing is performed. The quality, such as the quality of the library of the DNA library and/or the quality of the sequencing of the DNA library, is based on the above findings. Specifically, the present invention includes the following aspects:

 A first aspect of the invention relates to a locked nucleic acid modified DNA fragment for high throughput sequencing, wherein the DNA fragment is selected from one, two or three of a linker, a PCR primer and a sequencing primer, characterized in that The linker, PCR primer and/or sequencing primer in the DNA fragment contains a locked nucleic acid.

In one embodiment of the invention, the high throughput sequencing refers to SOLEXA sequencing. One, .

 In another embodiment of the invention, the DNA fragment is a PCR primer.

 In another embodiment of the invention, the DNA fragment is a sequencing primer.

 In another embodiment of the invention, the DNA fragment is a linker and a PCR primer.

 In another embodiment of the invention, the DNA fragment is a PCR primer and a sequencing primer. In another embodiment of the invention, the DNA fragment is a linker, a PCR primer, and a sequencing primer.

 A DNA fragment according to any one of the first aspects of the present invention, wherein the lock nucleic acid modification contained in the linker is located at a 5, end of the linker near the linker F chain, and/or near the 3 end of the linker R chain; preferably, said The 5th end near the F chain of the linker is located at the 2nd to 5th (for example, 2nd, 3rd, 4th, 5th) nucleotides of the F chain 5, and the 3rd end of the R chain near the linker refers to Located at the 2nd to 5th (for example, 2nd, 3rd, 4th, 5th) nucleotides of the R chain 3, preferably, the number of locked nucleic acids in the F chain or R chain of the linker is 1 to 3 (for example, 1, 2, 3).

 In one embodiment of the invention, the locked nucleic acid contained in the linker is located at the 5th nucleotide of the terminal F chain 5, and is located at the 3rd nucleotide of the end of the linker R chain 3, The number of locked nucleic acids in the F chain and the R chain of the linker is one.

 In a specific embodiment of the invention, the sequence of the linker F chain is the sequence of SEQ ID NO: 3; in a specific embodiment of the invention, the sequence of the linker R chain is SEQ ID NO: 4 The sequence shown.

 A DNA fragment according to any one of the first aspects of the present invention, wherein, in the PCR primer, the sense primer (upstream primer) contains a locked nucleic acid located near the 5th end of the PCR primer, preferably, the near 5 is Refers to the 2nd to 5th (eg, 2nd, 3rd, 4th, 5th) nucleotides near the 5th end of the primer; and/or the antisense primer (downstream primer) contains the locked nucleic acid located near the PCR primer 3, the end, preferably, the near 3, the end refers to the 2nd to 5th (for example, 2nd, 3rd, 4th, 5th) nucleotides located near the 3rd end of the primer; further preferably, The number of locked nucleic acids in the PCR primer is 1 to 3 (for example, 1, 2, 3).

 In an embodiment of the present invention, the sense primer refers to a PCR primer for amplifying a coding strand in genomic DNA, and in one embodiment of the present invention, a partial sequence thereof is identical to a fixed sequence P5 on a chip. PCR primers.

 In a particular embodiment of the invention, the PCR sequence of the partial sequence and the immobilized sequence P5 on the chip refers to a PCR Primer PE 1.0 primer.

 In one embodiment of the present invention, the locked nucleic acid contained in the PCR primer is located at the 3rd nucleotide of the PCR Primer PE 1.0 primer near the 5th end, and the number of the locked nucleic acid is 1.

 In an embodiment of the invention, the antisense primer refers to a PCR primer for amplifying a template strand in genomic DNA, and in one embodiment of the invention, a partial sequence thereof and a fixed sequence on the chip P7- The resulting PCR primers.

In a specific embodiment of the present invention, the PCR primer that binds to the immobilized sequence P7 on the chip comprises a tag sequence for distinguishing different sequencing results, thus the on-chip The number of libraries that are mixed and sequenced is related. In a specific embodiment of the present invention, four different libraries are mixed and sequenced, so the number of the tag sequences is four, so the PCR primers that bind to the immobilized sequence P7 on the chip The number is four.

 In an embodiment of the invention, the tag consists of A/T/C/G, which serves to identify different libraries, allowing different libraries to be mixed and sequenced to take full advantage of sequencing throughput. In a particular embodiment of the invention, the label has a length of 8 nt, such as AGAGACTT, GCGAGGCC. AGATCTCT or TAGAGAGC. During library construction, tags can be introduced by ligation tag ligation or PCR. In a specific embodiment of the invention, PCR primers are used to introduce tags using PCR, allowing different libraries to be labeled with different markers for sequencing.

 In a specific embodiment of the present invention, the PCR primer of the partial sequence and the immobilized sequence P7 on the chip is a PCR Primer PE2.0 primer, for example, PCR Primer PE2.0 primers A, B, C. D.

 In a specific embodiment of the present invention, the locked nucleic acid contained in the PCR primer is located at the 3rd nucleotide of the 3' end of the PCR Primer PE2.0 primer, and the number of the locked nucleic acid is 1.

 In a specific embodiment of the invention, the sequence of the PCR primer is the sequence shown in SEQ ID NO: 6.

 In a specific embodiment of the invention, the sequence of the PCR primer is the sequence shown in SEQ ID NOs: 11-14.

 According to the DNA fragment of any one of the first aspects of the present invention, the locked nucleic acid contained in the sequencing primer is located near the 3' end of the sequencing primer; preferably, the 3 end of the sequencing primer is located near the sequencing primer. At the 2nd to 5th (for example, 2nd, 3rd, 4th, 5th) nucleotides of the terminal; preferably, the number of locked nucleic acids in the sequencing primer is 1 to 3 (for example, 1, 2, 3) )).

 In an embodiment of the invention, the sequencing primer is a Read2 sequencing primer.

 In one embodiment of the present invention, the locked nucleic acid contained in the sequencing primer is located at the 2nd and 4th nucleotides of the sequencing primer near the 3' end, and the number of locked nucleic acids in the sequencing primer is 2.

 In a specific embodiment of the invention, the sequence of the sequencing primer is the sequence shown in SEQ ID NO: 16.

 A second aspect of the invention relates to a composition comprising the DNA fragment of any of the first aspects of the invention. A third aspect of the invention relates to a method for constructing a DNA library, the method comprising the step of performing a nucleic acid modification on a DNA fragment, the DNA fragment being a linker and/or a PCR primer, the lock nucleic acid modification being a linker in the DNA fragment and / or PCR primers contain a locked nucleic acid; preferably,

 The lock nucleic acid contained in the linker is located at the 5th end of the linker F chain, and/or 3 end of the linker R chain; preferably, the 5 end of the link F chain is located at the F chain 5 At the 2nd to 5th (for example, 2nd, 3rd, 4th, and 5th) nucleotides of the end, the 3rd end of the R chain near the linker is located at the 2nd to 5th of the R chain 3, for example, 2, 3, 4, 5) nucleotides; preferably, the number of locked nucleic acids in the F chain or R chain of the linker is 1-3 (for example, 1, 2, 3);

In the PCR primer, the sense primer contains a locked nucleic acid located near the PCR primer 5, , , , 3, 4, 5) nucleotides; and/or antisense primers contain a locked nucleic acid located near the 3' end of the PCR primer, preferably, close to 3, the end refers to the primer 2 to 5 (for example, 2, 3, 4, 5) nucleotides near the 3'end; further preferably, the number of locked nucleic acids in the PCR primer is 1 to 3 (for example, 1, 2, 3).

 In one embodiment of the invention, the locked nucleic acid contained in the linker is located at the 5th nucleotide of the F chain 5, and is located at the 3rd nucleotide of the R chain 3, the F chain The number of locked nucleic acids in the R chain and each of the R chains is one.

 In a specific embodiment of the invention, the sequence of the linker F chain is the sequence of SEQ ID NO: 3; in a specific embodiment of the invention, the sequence of the linker R chain is SEQ ID NO: 4 The sequence shown.

 In an embodiment of the present invention, the sense primer refers to a PCR primer for amplifying a coding strand in genomic DNA, and in one embodiment of the present invention, a partial sequence thereof is identical to a fixed sequence P5 on a chip. PCR primers.

 In a specific embodiment of the invention, the PCR primer that binds the partial sequence to the immobilized sequence P5 on the chip refers to a PCR Primer PE 1.0 primer.

 In one embodiment of the present invention, the locked nucleic acid contained in the PCR primer is located at the 3rd nucleotide of the PCR Primer PE 1.0 primer near the 5th end, and the number of the locked nucleic acid is 1.

 In an embodiment of the invention, the antisense primer refers to a PCR primer for amplifying a template strand in genomic DNA, and in one embodiment of the invention, a partial sequence thereof and a fixed sequence on the chip P7- The resulting PCR primers.

 In a particular embodiment of the invention, the PCR primers that bind to the immobilized sequence P7 on the chip comprise a tag sequence for distinguishing between different sequencing results, such that the binding to the immobilized sequence P7 on the chip The number of PCR primers is the same as the number of tag sequences. In a specific embodiment of the present invention, the number of the tag sequences is four, and therefore the number of the PCR primers combined with the immobilized sequence P7 on the chip is four.

 The number of tag sequences and positions in the PCR primers are well known in the art.

 In a specific embodiment of the present invention, the PCR primer of the partial sequence and the immobilized sequence P7 on the chip is a PCR Primer PE2.0 primer, for example, PCR Primer PE2.0 primers A, B, C. D.

 In a specific embodiment of the present invention, the locked nucleic acid contained in the PCR primer is located at the 3rd nucleotide of the 3' end of the PCR Primer PE2.0 primer, and the number of the locked nucleic acid is 1.

 In a specific embodiment of the invention, the sequence of the PCR primer is the sequence shown in SEQ ID NO: 6.

In a specific embodiment of the invention, the sequence of the PCR primer is the sequence shown in SEQ ID NOs: 11-14. A fourth aspect of the invention relates to a method of sequencing a DNA library, the method comprising modifying with a locked nucleic acid , ,

Ground,

 The locked nucleic acid contained in the sequencing primer is located near the 3 end of the sequencing primer; preferably, the 3 terminal near the sequencing primer refers to the 2nd to 5th of the sequencing primer near the 3rd end (for example, the 2nd and the 3rd) 4, 5) nucleotides; preferably, the number of locked nucleic acids in the sequencing primer is 1-3 (for example, 1, 2, 3).

 In an embodiment of the invention, the sequencing primer is a Read2 sequencing primer.

 In one embodiment of the present invention, the locked nucleic acid contained in the sequencing primer is located at the 2nd and 4th nucleotides of the sequencing primer near the 3' end, and the number of locked nucleic acids in the sequencing primer is 2.

 In a specific embodiment of the invention, the sequence of the sequencing primer is the sequence shown in SEQ ID NO: 16. A fifth aspect of the present invention relates to a high-throughput sequencing method, which comprises a method for constructing a DNA library and a method for sequencing a DNA library, the method for constructing the DNA library according to any one of the third aspect of the present invention, The sequencing method of the DNA library is the sequencing method according to any one of the fourth aspects of the invention. A sixth aspect of the invention relates to the use of a DNA fragment according to any of the first aspects of the invention in high throughput sequencing, construction of a DNA library or sequencing of a DNA library.

 In one embodiment of the invention, the high throughput sequencing refers to SOLEXA sequencing. The invention is further described below.

 In the present invention, the high-throughput sequencing is also called "Next-generation" sequencing technology, in order to be capable of paralleling hundreds of thousands to millions of DNA molecules at a time. Sequence determination is a marker, and high-throughput sequencing makes it possible to perform detailed analysis of the transcriptome and genome of a species, so it is also known as deep sequencing, including but not limited to: massively parallel signatures Massively Parallel Signature Sequencing (MPSS). Polymerase cloning (POLony Sequencing), 454 pyrosequencing, Illumina (Solexa) sequencing, ABI SOLiD sequencing, Ion semiconductor sequencing, DNA nanospheres DNA nanoball sequencing, Helicos' single-molecule DNA sequencing technology, etc., are preferably synthetic sequencing, such as SOLEXA sequencing and various sequencing technologies developed based on Solexa sequencing.

In the present invention, the SOLEXA sequencing belongs to the next-generation sequencing technology developed by SOLICA, and the core idea is to sequence while synthesizing. That is, when a new DNA complementary strand is generated, either the added dNTP catalyzes the substrate to catalyze the fluorescence by enzymatic cascade reaction, or directly adds the fluorescently labeled dNTP or semi-degenerate primer, and releases when the synthetic strand is synthesized or linked to form a complementary strand. Fluorescent signal. Complementary strand sequence information is obtained by capturing the optical signal and transforming it into a sequencing peak (Mardis ER (2008). x ,. u .

387-402). The SOLEXA test includes sequencing of DNA samples and sequencing of RNA samples. Depending on the sequencing method, SOLEXA sequencing can be divided into single-end sequencing (Single-read Sequencing) and double-ended sequencing (Paired-end Sequencing and Mate-pair Sequencing). In an embodiment of the invention, the SOLEXA sequencing method is a Paired-end sequencing method.

 In the present invention, the locked nucleic acid refers to a synthetic antisense oligonucleotide, which is a special bicyclic nucleotide derivative and also belongs to a kind of nucleotide. The 2,-oxygen and 4,-carbon of the ribose ring (β-D-ribofuranosyl) of the nucleotide residue form a fluorenylene linkage by shrinkage (see Formula I, where B is a base), and the structure contains One or more 2'-0,4'-C-arylene-β-D-ribofuranoic acid monomers, the VO position and the 4'-C position of ribose form an oxy-indenylene bridge through different shrinkage, a sulfinylene bridge or an amine sulfhydryl bridge, which is connected in a ring shape. This ring bridge locks the N-form of the furanose C3'-endotype, reduces the flexibility of the ribose structure, and increases the local structure of the phosphate skeleton.

在本发明中， 所述锁核酸修饰是指在 DNA片段中的核苷酸被具有相同碱 基的锁核酸替代。

In the present invention, the locked nucleic acid modification means that the nucleotide in the DNA fragment is replaced by a locked nucleic acid having the same base.

 In the present invention, the DNA library refers to a library prepared by extracting genomic DNA from a cell, then breaking it to a size of about 100-1000 bp, and then ligating the linker to the fragment and PCR-amplifying it. The library is used for high throughput sequencing, such as SOLEXA sequencing.

 In the present invention, the construction of the DNA library refers to a process from the extraction of DNA in a cell to the obtaining of a DNA library.

 In the present invention, the sequencing of the DNA library refers to a process of sequencing the obtained DNA library to obtain a nucleotide sequence of each fragment in the library.

 In the present invention, the DNA fragment refers to a small DNA fragment required for high-throughput sequencing such as SOLEXA sequencing, including a linker, a PCR primer, and a sequencing primer.

 In the present invention, the genomic DNA fragment refers to a fragment obtained by disrupting the extracted genomic DNA.

 In the present invention, the adaptor (Adapter) is used in high-throughput sequencing, particularly in SOLEXA sequencing, specifically to add a "Y"-type double-stranded DNA fragment at the end of the interrupted genomic DNA fragment, which is A "Y" type double-stranded DNA fragment synthesized by annealing the F chain and the R chain. The function of the linker is to add a known sequence to design the corresponding primer for PCR.

In the present invention, the PCR primer is used in high-throughput sequencing, particularly in SOLEXA sequencing, w.

 In the present invention, the sequencing primers are used in high-throughput sequencing, particularly SOLEXA sequencing, and specifically refer to primers for sequencing a constructed DNA library.

 In the present invention, the F chain of the linker refers to a forward oligonucleotide chain of a double-linker, wherein the 5, end sequence is complementary to the R chain 3, the end sequence, forming a Y-type double-stranded fragment, 5, After phosphorylation, it can be linked to the 3, end A of the genomic DNA fragment after addition of A.

 In the present invention, the 3' end sequence of the R chain of the linker is complementary to the F chain 5, the end sequence to form a Y-type double-stranded fragment, and the 3' end may be ligated to the 5' end of the genomic DNA fragment.

 In the present invention, the 5, or 3, terminal near the DNA fragment is located within a third of the length of the 5, or 3, end of the fragment.

 In the present invention, the n-th nucleotide located at the 5, or 3, end of a DNA fragment is calculated from the first nucleotide of the 5, or 3, end of the fragment. The position of the nth nucleotide.

 In the present invention, the flowcell refers to a sequencing chip to which a single-stranded oligonucleotide sequence is attached.

 In the present invention, the fixed sequence P7 refers to a binding sequence on a flowcell, and a 5, end sequence of a sequence sequence of a SBS process template.

 In the present invention, the fixed sequence P5 refers to a binding sequence on a flowcell.

 In the present invention, the sense primer is also referred to as an upstream primer, and refers to a primer which is identical to the 5' end sequence of the coding strand in the DNA fragment to be amplified.

 In the present invention, the antisense primer, also referred to as a downstream primer, refers to a primer complementary to the 3, end sequence of the coding strand in the DNA fragment to be amplified.

 In the present invention, the Readl sequencing primer refers to a sequencing primer used for synthesizing a read library 5, a terminal sequence at the time of double-end sequencing.

 In the present invention, the Read2 sequencing primer refers to a sequencing primer used for synthesizing the read library 3, the terminal sequence, at the time of double-end sequencing.

 In the present invention, the nucleotide includes deoxyribonucleotides, ribonucleotides, and also includes a locked nucleic acid.

In the present invention, the position of the locked nucleic acid means that the deoxyribonucleotide at that place is replaced by a corresponding locked nucleic acid, that is, by a locked nucleic acid containing the same base. In one embodiment of the present invention, a library of LNA-modified linkers and PCR primers is separately constructed, and a library of commonly used DNA small fragments that have not been modified by LNA is used, and after successful library preparation, solexa high-throughput sequencing is performed, wherein The LNA-modified library was subjected to LNA-modified supernatant sequencing primers, and the LNA-modified library was subjected to sequencing primers provided by Illumina, and the results obtained were compared to verify the stability, reproducibility and true reliability of the present invention. After selecting the same sample and performing multiple experiments, it is proved that the data obtained by the present invention is authentic, and the quality of the library modified by LNA and the quality of sequencing are improved compared with the data of the prior art. The invention combines the LNA modification technology with the high-throughput sequencing technology, and improves the thermal stability of the DNA fragment and the stability against the enzyme degradation by performing LNA modification on the linker, PCR primer and/or sequencing primer involved in SOLEXA sequencing. Sex, which activates RNase H, which reduces DNA dimer production and improves ligation and PCR efficiency.

 At the same time, the present invention optimizes the LNA modification site, and adopts different strategies for modification of different DNA fragments, thereby further improving the effect of LNA modification. For the LNA modification of the linker, the modification site is located at the 5th end of the F chain of the linker and at the 3rd end of the R chain, which can improve the efficiency of the joint annealing and improve the connection of the linker with the target fragment which is reacted with "A" reaction. To make the binding of the linker and the PCR primer more efficient, specific and sensitive; for the LNA modification of the PCR primer, the LNA modification site of the common PCR Primer PE 1.0 primer is close to the 5th end, which can improve the binding efficiency with the p5 sequence, PCR Primer PE2.0 primers (A, B, C, D) have LNA modification sites close to the 3' end, which improves the binding efficiency to the p7 sequence, and ultimately makes the sequence of interest and the immobilization sequence more stable; LNA for sequencing primers Modification, using double-modification of sequencing primers at the 3' end, can improve the stability, specificity and sensitivity of sequencing primers, thereby improving the quality of the entire sequencing Run (single-on-sequence sequencing reaction).

 The present invention performs parallel analysis on the sequencing data of the LNA modified library and the non-LNA modified library, and evaluates the sequencing quality by sequencing the base quality value (Q30%), the sequencing error rate, the GC content, the joint contamination, and the genomic alignment ratio. Library quality was evaluated by GC distribution, gene coverage, and the like. By comparison, it was found that the library modified with LNA was better than the library without LNA modification, both in library quality and in sequencing quality. DRAWINGS

 Figure 1: LNA modification protocol for SOLEXA sequencing;

 Figure 2: Schematic diagram of the preparation process of the DNA PE index library;

 Figure 3A: Non-LNA modified library sequencing results fq mass value q30 box plot, the abscissa is the number of cycles of PE90, the ordinate is Q30% (Q-64>=30, the percentage of qualified bases); box plot The drawing is to sort all the tiles (cells) Q30% in each cycle, and draw 5 points into a box plot: highest, lowest, median, quarter, three-quarters.

 Figure 3B: LNA modified library sequencing results fq mass value q30 box plot, the abscissa is the number of cycles of PE90, the ordinate is Q30% (Q-64>=30, the percentage of qualified bases); In each cycle, the Q30% of all tiles are sorted, and 5 points are drawn into a box plot: highest, lowest, median, quarter, and three-quarters.

 Figure 4A: Non-LNA modified library sequencing mass distribution map, the abscissa is the number of cycles of PE90, the ordinate is the base mass value corresponding to each cycle, the color represents a different percentage, white: 0, green: 10%, yellow: 30%, red: 50%, deep red: 70%, black: 100%;

Figure 4B: LNA modified library sequencing mass distribution map, the abscissa is the number of cycles of PE90, the ordinate is the base mass value corresponding to each cycle, the color represents a different percentage, white: 0, green: 10%, yellow: 30 %, red: 50%, dark red: 70%, black: 100%; , ,

The number of cycles, the ordinate is the error rate (number of bases per cycle error / number of bases in all cycles);

 Figure 5Β: The base error rate distribution of the LNA modified library sequencing results, the abscissa is the number of cycles of ΡΕ90, and the ordinate is the error rate (number of bases per cycle error / number of bases in all cycles);

 Figure 6Α: Non-LNA modified library sequencing results GC content distribution map, the abscissa is the GC content of each window on the statistical Acinetobacter reference sequence, and the ordinate is the number of times of comparison to each window;

 Figure 6: LNA modified library sequencing results GC content distribution map, the abscissa is the GC content of each window on the statistical Acinetobacter reference sequence, and the ordinate is the number of times of comparison to each window;

 Figure 7: Gene coverage map of library sequencing results, the abscissa is the number of coverages per base, and the ordinate is the number of bases; where the light curve (lower peak curve) indicates a non-LNA modified library, dark The curve (higher curve of the peak) represents the LNA modified library;

 Figure 8: Aglient 2100 results for unannealed joint annealing;

 Figure 8B: Aglient 2100 results for LNA-modified joint annealing;

 Figure 9A: Library sequencing results using unmodified read2 sequencing primers fq mass value q30 box plot, abscissa is the number of cycles of PE90, ordinate is Q30% (Q-64>=30, the percentage of qualified bases) The box plot is drawn by sorting Q30% of all tiles in each cycle and plotting 5 points as a box plot: highest, lowest, median, quarter, three-quarters;

 Figure 9B: Library sequencing results using LNA modified read2 sequencing primers fq mass value q30 box plot, abscissa is the number of cycles of PE90, ordinate is Q30% (Q-64>=30, the percentage of qualified bases) The box plot is drawn in such a way that Q30% of all tiles are sorted and 5 points are drawn into a box plot: highest, lowest, median, quarter, three quarters.

 detailed description

 The embodiments of the present invention are described in detail below with reference to the accompanying drawings, but the invention is to be construed as illustrative only. If no specific conditions are specified in the examples, they are carried out according to the general conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not indicate the manufacturer, they are all conventional products that can be obtained commercially. The following sequencing was performed using an Illumina Hiseq 2000 sequencer, and instructions were performed according to the instructions unless otherwise noted.

 In the following examples, unmodified DNA fragments were synthesized by Invitrogen, and LNA-modified DNA fragments were synthesized by Exiqon. Example 1 SOLEXA High-throughput Sequencing Method

Acinetobacter genomic DNA (genome ~ 3.6M, GC content 40.4%) was extracted as a template, about 30μg, the starting amount of each library was 3μ _§ , the Covaris S2 was used to break the main band 350bp, and 8 inserts were constructed in parallel. A 350 bp DNA PE inde library, 4 of which used LNA-modified adapters and PCR primers, and the other 4 libraries used LNA-free linkers and PCR primers. After the library preparation was completed, high-throughput sequencing of solexa (SBS-sequencing by synthesis) was carried out, in which the LNA-modified library was sequenced with LNA-modified sequencing primer read 2, and the library without LNA modification used the sequencing primer provided by Illumina. 350bp DNA PE index library preparation operation - See Figure 1, Figure 2. Specific steps are as follows:

 1. Genomic DNA interruption

Take an amount of Acinetobacter genomic 3μ _§, using the Covaris S2 (Covaris Corporation) interrupt to the main band detected 350bp _o interrupted qualified, QIAquick PCR Purification Kit (QIAGEN) and purified by broken product, dissolved in 32μ1 EB buffer.

 2, end repair

 The above procedure yielded 30 μΐ of eluted DNA, and 45 μl of water, 10 μΐ 10× End Repair Buffer (ENZYMATICS), 4 μΐ lOmM dNTP Mix (NEB), 5 μΐ T4 DNA Polymerase (ENZYMATICS), 1 μΐ Klenow were sequentially added to the reaction system. DNA Polymerase (ENZYMATICS), 5 μΐ T4 PNK (ENZYMATICS), the total reaction system was 100 μΐ, and the reaction tube was placed at 20 ° C for 30 min.

 After completion of the reaction, the terminal repair product was purified by QIAquick PCR Purification Kit (QIAGEN) and dissolved in 34 μl EB buffer.

 3, plus A

 The above procedure yielded 32 μΐ Eluted DNA, and 5μ1 10X blue Buffer ( ENZYMATICS ), 10 μΐ 1 mM dATP ( ENZYMATICS ), 3 μΐ Klenow exo (3' to 5' exo minus) (ENZYMATICS ) were added to the reaction system in turn. The system was 50 μl and the reaction tube was placed at 37. C reaction 30 min.

 After completion of the reaction, the product was purified by MinElute PCR Purification Kit (QIAGEN) and dissolved in 12 μl of EB buffer.

 4, add connector

 The above procedure results in a ΙΟμΙ addition product, and 25μ1 2x Rapid T4 DNA Ligase Buffer (ENZYMATICS), 10 μΐ PE Adapter Oligo Mix (PE connector mixture, 20 μΜ) or ΙΟμΙ LNA modified PE Adapter Oligo Μΐχ (20 μΜ) are sequentially added to the reaction system. 5μ1 T4 DNA Ligase (ENZYMATICS), the total reaction system was 50μ1, and the reaction tube was placed at -20 °C for 15 min.

 Unmodified PE joint:

 Adapter— F-1:

 5 Thos/GA TCGGAA GA GCA CA CGTCTGAA CTCCA GTCA C3 ' (where 5, Phos indicates 5, phosphorylation) (SEQ ID NO: 1)

 Adapter— R-1:

 5'TACACTCTTTCCCTACACGACGCTCTTCCGATCT3' (SEQ ID NO:

2)

LNA modified PE joint:

 Adapter— F-1:

5 Thos/GA TCGGAA GA GCA CA CGTCTGAA CTCCA GTCA C3 ' (where 5, Phos indicates 5, phosphorylation) (SEQ ID NO: 3) 5'TACACTCTTTCCCTACACGACGCTCTTCCGATCT3' (SEQ ID NO: 4)

 The underlined part is the LNA modification site of the linker, which represents the LNA modified G and T, respectively. The LNA modification site is close to the 5th end of the F chain, the R chain 3, and the end, which can improve the joint annealing efficiency and improve The ligation of the linker with the target fragment reacted with "addition of A" makes the binding of the linker and the PCR primer more efficient, specific and sensitive.

 After the reaction was completed, the ligated product was purified by QIAquick PCR Purification Kit (QIAGEN) and dissolved in 32 μl of EB buffer.

 5, the connection product glue purification

 The ligated product prepared in the step 4 was prepared, and a 2% agarose gel was prepared, and 50 bp DNA Ladder (NEB) was selected, and 120 rpm was electrophoresed for 60 minutes. The gelation was recovered, and the range of the gel was determined according to the size of the linker and the desired fragment size. The cut pieces were recovered by QIAquick Gel Extraction Kit (QIAGEN) and finally dissolved in 23μ1 EB buffer.

 6, PCR amplification

 The above procedure yielded a 23 μl ligation product, and 25 μΐ of Phusion® High-Fidelity DNA Polymerase (NEB), 1 μΐ PCR Primer PE 1.0 ( ΙΟμΜ ) or 1 μΐ LNA modified PCR Primer PE 1.0 ( 10 μΜ ), 1 was sequentially added to the reaction system. Μΐ PCR Primer PE 2.0 ( 10μΜ ) or 1 μΐ LNA modified PCR Primer PE 2.0 (10μΜ), total reaction system 50μ1. The reaction was carried out on the PCR machine according to the following procedure:

 a. 98 ° C, 30 s;

 b. The following procedure 10 cycles:

 98 ° C, 10 s; 65 ° C, 30; 72 ° C, 30 s;

 c. 72 ° C, 5 min;

 d. 4 ° C, maintained.

 The PCR Primer PE 1.0 primer (5, end primer, upstream primer) sequence is:

CGCTCTTCCGATCT3 (SEQ ID NO: 5)

 The LNA-modified PCR Primer PE 1.0 primer sequence is:

CGCTCTTCCGATCT3 (SEQ ID NO: 6)

 The underlined part is the LNA modification site of the linker, indicating the LNA modified T.

 The four unmodified PCR Primer PE 2.0 primers (3, end primers, downstream primers) sequence are:

 PCR _primer_A:

 5'CAAGCAGAAGACGGCATACGAGATAGAGACTTGTGACTGGAGTTCAGA CGTGTGCTCTTCCGA TCT3 ' (SEQ ID NO : 7)

PCR _primer_B: "

ACGTGTGCTCTTCCGA TCT3 ' (SEQ ID NO : 8)

 PCR _primer_C:

 5'CAAGCAGAAGACGGCATACGAGATAGATCTCTGTGACTGGAGTTCAGA CGTGTGCTCTTCCGA TCT3 ' (SEQ ID NO : 9)

 PCR _primer_D:

 5'CAAGCAGAAGACGGCATACGAGATTAGAGAGCGTGACTGGAGTTCAGA CGTGTGCTCTTCCGA TCT3 ' (SEQ ID NO : 10)

The four LNA-modified PCR Primer PE 2.0 primer sequences are:

 PCR _primer_A:

 5'CAAGCAGAAGACGGCATACGAGATAGAGACTTGTGACTGGAGTTCAGA CGTGTGCTCTTCCGATCT3 ' (SEQ ID NO : 11)

 PCR _primer_B:

 5'CAAGCAGAAGACGGCATACGAGATGCGAGGCCGTGACTGGAGTTCAG ACGTGTGCTCTTCCGATCT3 ' (SEQ ID NO : 12)

 PCR _primer_C:

 5'CAAGCAGAAGACGGCATACGAGATAGATCTCTGTGACTGGAGTTCAGA CGTGTGCTCTTCCGATCT3 ' (SEQ ID NO : 13)

 PCR _primer_D:

 5'CAAGCAGAAGACGGCATACGAGATTAGAGAGCGTGACTGGAGTTCAGA CGTGTGCTCTTCCGATCT3 ' (SEQ ID NO : 14)

 The LNA modification sites of the underlined linker represent the LNA modified T, respectively.

 Since the 5' end portion of the common PCR Primer ΡΕ 1.0 primer coincides with the fixed sequence P5 on the flowcell, the partial sequence of the PCR Primer PE 2.0 primer is identical to the fixed sequence P7 on the flowcell, underlined. For the LNA modification site of the PCR primer, the LNA modification site of the common PCR Primer PE 1.0 primer is close to the 5, and the binding efficiency of the P5 complementary sequence (DNA library template strand) can be improved. PCR Primer PE2.0 primer (A) The LNA modification site of B, C, D) is close to the 3, and the binding efficiency of the P7 complementary sequence (DNA library coding strand) can be improved, and finally the target sequence is more stably bound to the fixed sequence.

 Wherein the binding sequence in the fixed sequence P5 is: AATGATACGGCGACCACCGA ( SEQ ID NO: 19);

 The binding sequence in the fixed sequence P7 is: CAAGCAGAAGACGGCATACGA (SEQ ID NO: 20).

 7. PCR product gel purification

Take the PCR amplification product obtained in step 6. Prepare 2% agarose gel, select 50 bp DNA Ladder, 120v electrophoresis for 60min, and cut the gel. The range of gelation is determined by the size of the linker and the desired target fragment. The cut rubber pieces are recovered by QIAquick Gel Extraction Kit and finally dissolved in 25μ1 ΕΒ u

 8. After the library preparation is completed, solexa high-throughput sequencing is performed.

 Four LNA modifications and four non-LNA modified libraries were constructed in parallel according to the above method. After quality control, sequencing was performed, and the Illumina Hiseq2000 sequencer was used for sequencing. Four LNA-modified libraries were read with LNA-modified Read 2 sequencing primers, 4 Non-LNA modified libraries used Read 2 sequencing primers provided by Illumina.

 Readl sequencing primers:

 5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT3' (SEQ ID NO: 17) Inde Sequencing Primers:

 5' GATCGGAAGAGCACACGTCTGAACTCCAGTCAC3' ( SEQ ID NO:

18)

 Unmodified Read 2 sequencing primers:

 5, GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT3, (SEQ ID NO: 15) LNA-modified Read 2 sequencing primer:

 5 'GTGA CTGGA GTTCA GA CGTGTGCTCTTCCGA TCT3 ' (SEQ ID NO: 16) wherein the underlined portion is the sequencing primer LNA modification site, which represents LNA-modified A and (:. Because of high-throughput sequencing, the time is long in the sequencing process. Primers are easily degraded, which affects the quality of sequencing. Therefore, it is very important to modify the sequencing primers, especially at the end of the 3rd end. The present invention double-modifies the sequencing primers at the 3' end, which means to improve the sequencing primers. Stability, specificity and sensitivity to improve the quality of the entire sequencing run. Example 2 Analysis of SOLEXA high-throughput sequencing results

 Parallel analysis of the LNA modified library obtained in Example 1 and the non-LNA modified library data (ie, sequencing result data), mainly including sequencing base quality values after sequencing, sequencing error rate, %GC content, joint contamination, genome The comparison rate to evaluate the quality of the sequencing; and further analysis of the basic information of the data, GC distribution, gene coverage, etc., to evaluate the library quality. By comparison, it can be found that the library modified with LNA is generally better than the library without LNA modification, regardless of the quality of the library or the quality of the sequencing. (The following data results were obtained by the software included in Hiseq2000 sequencing except that the sequencing base quality values were obtained, and the rest were calculated by SOAP comparison analysis software, wherein Figures 3A and 3B are the average results of the four libraries)

 The results of the sequencing information analysis are shown in Tables 1 and 2, wherein the results in Tables 1 and 2 are the results of sequencing analysis of a set of parallel constructed libraries. Table 1 Sequencing information analysis results of unmodified library

_: 总读段数

_: Total number of readings

 1,733,3 total alkali amount read length (%Phasing) 0.141

( Total 311,994,720 90; 90

 (Length) Predetermined Phase Rate 0.397; Reads) 04 ( TotalBases )

 (%prephasing) 0.248

%Q20 97.94; %Q30 93.50; 87.13 GC content 41.40; error rate 0.12;

 93.03 (%GC) 41.35 (%ErrorRate) 0.43 Label (Index) Mismatch in AGAGAT index 97.6 Mismatch in index 2.3

 The ratio of the ratio of CT to 0 is 1.

 (%Index example (%Index

 Omismatch) lmismatch)

 The ratio is 99.85; the paired reads are only 96.6 inserts large 320 insert size error -12/+15 (%Align) 97.96 ratio is small ( Insert Size

 ( %Uniqpair) ( InsertSize ) SD)

Sequencing information analysis results of LNA modified library

表 1、表 2的数据结果由 Hiseq2000测序机器自带的软件自动得出，从表 1、 表 2可以看出： LNA修饰的文库， Q30%为 （96.77; 96.15 ) ; 而非 LNA修饰 的文库， ％Q30为： （93.50; 87.13 ) ; Q30% ( Q-64>=30, 即合格碱基的百分 比）越高表示质量值越好;不动杆菌的％0€实际含量是 40.4%，表 1中 LNA修 饰的文库％。<： ( 40.91; 40.84 )比非 LNA修饰的文库⁰ /oGC: ( 41.40; 41.35 ) 更靠近真实情况。错误率和比对率， 使用 LNA修饰的文库结果比对率更高， 错 误率更低， 具体数据对照表 1。

The data results in Table 1 and Table 2 are automatically obtained by the software provided by the Hiseq2000 sequencing machine. It can be seen from Table 1 and Table 2: LNA modified library, Q30% is (96.77; 96.15); non-LNA modified library , %Q30 is: (93.50; 87.13); Q30% (Q-64>=30, the percentage of qualified bases) indicates that the quality value is better; the actual content of Acinetobacter is 40.4%, Table % of the LNA modified library in 1 . <: ( 40.91; 40.84 ) is closer to the real situation than the non-LNA modified library ⁰ /oGC: ( 41.40; 41.35 ). The error rate and the comparison rate, the LNA modified library results in higher acknowledgment rate and lower error rate. The specific data are compared with Table 1.

Sequencing results fq mass value q30 box plot see Figure 3A, 3 B, Figure 3A is the sequencing result without LNA modification, Figure 3B is the LNA modified sequencing results, the abscissa is the number of cycles of PE90 (cycle), ordinate For Q30% (Q-64>=30, the percentage of qualified bases), it can be seen from the figure that the more concentrated the box plot is, the closer the value of Q30% is in each cycle, in the box plot. Value , . ,

The sequencing result is better than the unmodified sequencing result. The q30 box plot is concentrated, and the median value is also significantly higher. It is indicated that the base quality of the library modified with LNA is better than no modification.

 See Figure 4A, 4B for the sequencing quality distribution. Figure 4A shows the sequencing results without LNA modification. Figure 4B shows the sequencing results with LNA modification. The abscissa is the cycle number of PE90, and the ordinate is the base corresponding to each cycle. Base quality value, color represents a different percentage, white: 0, green: 10%, yellow: 30%, red: 50%, dark red: 70%, black: 100%, such as a certain quality value at a certain position Then, the mass value of the locus on the abscissa is 10% of the total mass value of the ordinate. The higher the mass value, the darker the color, the better the quality. It can be seen from Figures 4A and 4B that the LNA-modified library has a significantly better mass value distribution than the LNA-free library.

 See Figure 5A, 5B for the base error rate distribution. Figure 5A shows the sequencing result without LNA modification. Figure 5B shows the sequencing result of LNA modification. The data of the lower machine is compared with the reference genome. Two 32 bp are allowed. In the case of mismatch, select the segment (sss). If the first 32 bp is matched in the case of allowing 2 mismatches, use eland software to calculate, in the comparable reads, each cycle is wrong. Number of bases / number of bases in all cycles. The abscissa indicates the number of cycles of PE90, the ordinate is the number of bases per cycle error/the number of bases in all cycles. From the results, the LNA-modified library has a lower error rate than the unmodified library. many.

 See Figure 6A, 6B for GC content distribution, Figure 6A for sequencing results without LNA modification, Figure 6B for LNA-modified sequencing results, one window per 500 bp, and the abscissa for each window on the statistical Acinetobacter reference sequence The GC content, the ordinate is the number of times of comparison to each window, and the correlation analysis is performed to analyze the PCR preference of the GC. The more uniform the number of coverages, the lower the GC preference. From the comparison in the figure, the preference of GC was significantly reduced after LNA modification.

 The gene coverage distribution is shown in Figure 7. The abscissa is the number of times of coverage of each base, and the ordinate is the number of bases. The curve conforms to the Poisson distribution. The more concentrated the graph is on the central axis, the more random the coverage is. The figure shows that the coverage randomness is improved after the LNA modification. Table 3 Sequencing information of two sets of parallel library libraries

测序深度（Depth ) 72 74.1 68.3 74.1 覆盖度 ( Coverage ) 98.85% 98.82% 98.84% 98.84%

Depth of Depth (Depth) 72 74.1 68.3 74.1 Coverage 98.85% 98.82% 98.84% 98.84%

 Coverage ≥40χ

 ( coverage at least 40 x ) 0.943721 0.978885 0.93051 0.978784

 Coverage ≥60x

 ( coverage at least 60 x ) 0.745516 0.832488 0.681204 0.831284

 Coverage ≥80x

 ( coverage at least 80 x ) 0.340046 0.355042 0.267971 0.357859

 (Parts a and b refer to unmodified libraries, respectively, and groups A and B refer to LNA-modified texts, respectively.

As can be seen from the above table, the LNA-modified library increased the unique rate and map to genome rate, reduced the duplication rate, and the coverage and depth were better than those without the LNA modified library.

 It is thus confirmed that the LNA modified adapter, PCR primer, sequencing primer can be applied to high-throughput database sequencing technology, which can improve library quality, reduce invalid data, and increase sequencing accuracy. Sex. Example 3 Annealing Results of LNA Modified and Unmodified Linkers

 The F chain and the R chain (SEQ ID NO: 1 and SEQ ID NO: 2, respectively) of an unmodified hydrazine linker of equal volume and concentration of 100 μM were subjected to gradient joint annealing, and after annealing, diluted to 5 μΜ , using the Agilent 2100 for testing, the results are shown in Figure 8A.

 The F chain and the R chain (SEQ ID NO: 3 and SEQ ID NO: 4, respectively) of an LNA modified ΡΕ linker of equal volume and concentration of 100 μΜ were subjected to gradient joint annealing, and after annealing, diluted to 1 μΜ The test was carried out using an Agilent 2100, and the results are shown in Fig. 8B.

 It can be seen from Fig. 8A and Fig. 8B that the size of the synthesized double strands after annealing is 80 bp and 82 bp, respectively, and the proportion of the double-linked head synthesized by LNA modification is 60%, and the peak of the single chain is only one, and there is no modification. The ratio of the double link head is 57%, and the peak of the single chain has two.

 The results show that the concentration of the double-stranded and the annealing efficiency of the LNA-modified PE linker is higher than that of the unmodified joint. Example 4 Sequencing results using LNA-modified and unmodified sequencing primers

A set of parallel libraries was prepared according to the method of Example 1 and subjected to high-throughput sequencing of SOLEXA, except that when the linker was added, the linker used was a linker not modified by LNA (SEQ ID NO: 3 and SEQ ID NO: 4). When performing PCR amplification, the PCR primers used were PCR primers without LNA modification (SEQ ID NOS: 7-10), and only LNA-modified and unmodified sequencing primers were used for sequencing (SEQ ID NO: 15 ~ 16). The sequencing results are shown in Tables 4 and 5.

其中 RawClusters/Tile 表示每个 tile的 DNA簇（ clusters )数， 这里取了 所有 tile的中位数。 PFClusters/Tile 表示每个 tile经过 PF过滤之后的 DNA簇 数， PF是 Illumina的默认的过滤规则： 前 25个碱基里面只允许有一个碱基质量 不好， 不满足这个条件的 reads 就过滤。 ％PF : PFClusters/RawClusters FirstCyclelnt: 第一个循环的光强 。 ％Phasing: 反应滞后的概率 （当前循环数 中， 还在进行前 1个循环反应的 reads的比例）。 ％Prephasing: 反应超前的概率 (当前循环数中，已经在进行后 1个循环反应的 reads的比例）。％Q20( Q-64>=20 即合格碱基的百分比） ， ％Q30 ( Q-64>=30, 即合格碱基的百分比） 。

Where RawClusters/Tile represents the number of clusters of clusters per tile, where the median of all tiles is taken. PFClusters/Tile indicates the number of DNA clusters after each tile has been filtered by PF. PF is Illumina's default filter rule: Only one base in the first 25 bases is allowed to have a bad quality. Reads that do not meet this condition are filtered. %PF : PFClusters/RawClusters FirstCyclelnt: The light intensity of the first cycle. %Phasing: Probability of response lag (in the current number of cycles, the proportion of reads that are still in the previous cycle). %Prephasing: Probability of response advancement (the proportion of reads in the current cycle number that has already been processed in the last cycle). %Q20 (Q-64>=20 is the percentage of qualified bases), %Q30 (Q-64>=30, which is the percentage of qualified bases).

As can be seen from Tables 4 and 5, sequencing results using LNA-modified read2 sequencing primers , „

The quality of sequencing is better.

 9A and 9B are box results of sequencing results of fq mass value q30 using LNA-modified or unmodified sequencing primer read2, wherein the more concentrated the box plot, the closer the value of Q30% is in each cycle, the box The higher the median value in the graph, the better the base quality. As can be seen from the two figures, the sequencing result of the sequencing primer using the LNA modified read2 q30 box plot is more concentrated than the sequencing result of the unmodified sequencing primer read2 q30 box plot, the median value is also higher, indicating the use of LNA The modified sequencing primer read2 is better than the base of the unmodified sequencing primer read2.

 Comparing Table 2 with Table 4, Figure 9B and 3B, it can be seen that both Q20 and Q30 of Table 2 are higher than those of Table 4, while the q30 box diagram of Figure 3B is more concentrated than that of Figure 9B, and the median value is also Significantly improved. The results indicate that PCR primers and sequencing primers have better sequencing results than LSA-modified primers. Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand. Various modifications and alterations may be made to those details in light of the teachings of the invention, which are within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

Rights request  A lock nucleic acid-modified DNA fragment for high-throughput sequencing, wherein the DNA fragment is selected from one, two or three of a linker, a PCR primer, and a sequencing primer, characterized in that the DNA fragment is The linker, PCR primer and/or sequencing primer contains a locked nucleic acid. 2. The DNA fragment of claim 1, wherein the linker nucleic acid contained in the linker is located at the 5' end of the linker near the F chain, and/or near the 3' end of the R chain; preferably, the 5' end near the F chain It refers to the 2nd to 5th (for example, 2nd, 3rd, 4th, 5th) nucleotides located at the 5th end of the F chain, and the 3rd end near the R chain refers to the 2nd to the end of the R chain 3 5 (for example, 2, 3, 4, 5) nucleotides; preferably, the number of locked nucleic acids in the F chain or R chain is 1 to 3 (for example, 1, 2, 3) . 3. The DNA fragment according to claim 1, wherein the PCR primer comprises a lock nucleic acid contained in the sense primer located near the 5th end of the PCR primer, preferably, the near 5 is at the end of the primer near the 5th end. 2 to 5 (eg, 2, 3, 4, 5) nucleotides; and/or the antisense primer contains a locked nucleic acid located near the 3' end of the PCR primer, preferably, the proximity 3 The terminus refers to the 2nd to 5th (for example, 2nd, 3rd, 4th, 5th) nucleotides located near the 3rd end of the primer; further preferably, the number of locked nucleic acids in the PCR primer is 1~ 3 (for example, 1, 2, 3). 4. The DNA fragment of claim 1, wherein the sequencing primer comprises a locked nucleic acid located near the 3' end of the sequencing primer; preferably, the 3 terminus adjacent to the sequencing primer refers to the second of the sequencing primer near the 3' end. ~ 5 (for example, 2, 3, 4, 5) nucleotides; preferably, the number of locked nucleic acids in the sequencing primer is 1 to 3 (for example, 1, 2, 3). A composition comprising the DNA fragment of any one of claims 1-4. A method for constructing a DNA library, the method comprising the step of constructing a library using a DNA fragment modified with a locked nucleic acid, wherein the DNA fragment is a linker and/or a PCR primer, and the lock nucleic acid modification refers to a linker and a PCR in the DNA fragment. The primer contains a locked nucleic acid; preferably, The lock nucleic acid contained in the linker is located at the 5th end of the linker F chain, and/or 3 end of the linker R chain; preferably, the 5 end of the link F chain is located at the F chain 5 , the second 2 ~ 5 At the nucleotides (for example, 2, 3, 4, 5), the 3, end of the R chain near the linker is located at the 2nd to 5th nucleotides of the R chain 3, preferably; The number of locked nucleic acids in the F chain or the R chain of the linker is 1 to 3 (for example, 1, 2, 3);  Or, preferably,  In the PCR primer, the locked nucleic acid contained in the sense primer is located near the 5th end of the PCR primer, preferably, the close to 5, the end refers to the 2nd to 5th of the primer near the 5th end (for example, the second , 3, 4, 5) nucleotides; and/or the antisense primer contains a locked nucleic acid located near the 3' end of the PCR primer, preferably, the close to 3, the end is located at the primer close to 3 Further, the second to fifth (for example, the second, third, fourth, and fifth) nucleotides of the terminal; further preferably, the number of locked nucleic acids in the PCR primer is from 1 to 3 (for example, 1, 2, 3). 7. A sequencing method for a DNA library, the method comprising the step of sequencing using a locked nucleic acid-modified sequencing primer, wherein the locked nucleic acid modification comprises a sequencing primer comprising a locked nucleic acid; preferably, the sequencing primer comprises a locked nucleic acid Located at the 3' end of the sequencing primer; preferably, the 3 terminus near the sequencing primer refers to the 2nd to 5th (eg, 2nd, 3rd, 4th, 5th) nucleotides located near the 3rd end of the sequencing primer. Preferably, the number of locked nucleic acids in the sequencing primer is 1-3 (for example, 1, 2, 3). A high-throughput sequencing method comprising the construction of a DNA library and the sequencing of the DNA library, the method of constructing the DNA library is the construction method according to claim 6, the sequencing method of the DNA library is the method of claim 7. Sequencing method. 9. Use of a DNA fragment according to any one of claims 1 to 4 in high throughput sequencing, construction of a DNA library or sequencing of a DNA library.