Preparation method of sequencing library and kit for preparing sequencing library

Abstract

The invention provides a preparation method of a sequencing library and a kit for preparing the sequencing library. The preparation method comprises the following steps: (i) obtaining a nucleic acid fragment to be detected; (ii) adding a linker element pair and a cyclization element, and carrying out connection and cyclization reaction with the nucleic acid fragment to be detected; and (iii) carrying out extension reaction to obtain the sequencing library. The invention further provides a method for preparing the nucleic acid library to be detected through the single tube. According to the method, an original cyclization step is not needed, and library cyclization and DNB preparation are completed in the linker connection process; and the traditional three-step reaction is reduced to one-step independent reaction, so that the time is shortened, the steps are simplified, and the pollution is avoided.

Description

A method for preparing a sequencing library and a kit for preparing a sequencing library Technical Field

[0001] The present invention relates to the field of biological analysis and detection, and in particular to a method for preparing a sequencing library and a kit for preparing the sequencing library. Background Art

[0002] With the development of high-throughput gene sequencing technology, the cost of gene sequencing has dropped rapidly, but the cost of second-generation sequencing sample pre-processing - library preparation is still high and the process is still complicated. Pre-sequencing sample processing has become a bottleneck restricting the large-scale application of gene sequencing technology.

[0003] High-throughput sequencing library preparation requires multiple steps, including DNA fragmentation, end-repair, end-addition of A fragments, adapter ligation, and PCR. This complex, time-consuming, and labor-intensive process can delay research progress.

[0004] The DNBSEQ sequencer launched by BGI is based on the sequencing principle of DNB (DNA Nano Ball). The rolling circle linear amplification principle is used in the base signal amplification process, and its sequencing accuracy is better than the methods based on exponential amplification such as bridge (such as Illumina). The existing library preparation technology of the DNBSEQ platform includes DNA fragmentation, end repair and end A addition, adapter ligation, PCR, library circularization and DNB preparation. The whole process contains 6 steps. However, the library preparation method of this platform has an additional circular library preparation process compared to the library preparation methods of other platforms. Although its sequencing accuracy has been improved, its library preparation link is also more complicated, which increases the burden of sample pre-processing. In addition, the existing DNBSEQ library preparation method requires three independent steps of adapter ligation, circularization and DNB preparation. The biggest disadvantage of this method is that it has many steps, cumbersome operation and long time. In addition, due to the many steps of conventional library preparation and the low DNA utilization efficiency in some steps, such as the circularization step, a large amount of DNA input is required to meet the final library sequencing. For trace amounts of DNA, PCR is required to meet the requirements for the machine.

[0005] Summary of the Invention

[0006] In order to solve the technical problem of the lack of a rapid and efficient method for preparing a DNB library in the prior art, the present invention provides a method for preparing a sequencing library and a kit for preparing a sequencing library.

[0007] Based on existing technologies, the present invention optimizes the library construction method and develops an application system and method that can perform linker ligation, cyclization, and DNB preparation in a single reaction system. This greatly reduces the number of library preparation steps and improves the efficiency of DNA utilization. It can meet the needs of library construction for trace nucleic acids and provide a more flexible and rapid library construction method.

[0008] A first aspect of the present invention provides a method for preparing a sequencing library, comprising the following steps:

[0009] (i) obtaining a nucleic acid fragment to be detected;

[0010] (ii) adding a linker element pair and a cyclization element to undergo ligation and cyclization reactions with the nucleic acid fragment to be detected; wherein the linker element pair comprises a linker element A and a linker element B, wherein both the linker element A and the linker element B are composed of a first nucleotide chain and a second nucleotide chain, and the first nucleotide chain and the second nucleotide chain form a partially complementary double-stranded structure; the cyclization element is a single-stranded nucleic acid molecule having reverse-complementary nucleic acid sequences at both ends, and the middle sequence and 3' end sequence of the cyclization element are partially complementary to the linker element A and linker element B, respectively;

[0011] (iii) Perform an extension reaction to obtain a sequencing library.

[0012] As used herein, the terms "linker element A" and "linker element B" have no specific meanings; A and B are interchangeable. In each linker element, the 5' and 3' ends of the first nucleotide chain are partially complementary to the 3' and 5' ends of the second nucleotide chain, respectively. The terms "first" and "second" have no specific meanings, as long as they distinguish between two partially complementary nucleotide chains. When the linker element pair is ligated to the nucleic acid fragment to be tested, a ligation product with linker elements at both ends, i.e., a double-stranded DNA structure, is formed.

[0013] In some preferred embodiments, in step (ii), the linker element pair is connected to the two ends of the nucleic acid fragment to be tested to form a nucleic acid fragment with different linker elements at both ends (i.e., a double-stranded DNA structure), and then the nucleic acid fragment with different linker elements at both ends is complementary hybridized with the cyclization element to form a partially double-stranded circular nucleic acid structure.

[0014] Herein, the purpose of the cyclization element (or cyclization element C) being in a hairpin structure is to prevent the connection or complementation between the linker element and the cyclization element, and only allow the linker element to react with the nucleic acid fragment to be tested. The term "intermediate sequence" refers to the single-stranded loop sequence on the cyclization element except for the complementary sequences at the 5' end and 3' end. The cyclization element is in a hairpin structure at room temperature, and when the temperature rises to above 60°C, the hairpin structure opens to a linear structure. After the linker element and the nucleic acid fragment to be tested are correctly connected, at a temperature of 60-80°C, the ends of the double-stranded DNA structure of the connection product are denatured into single strands due to the increase in temperature. As a result, the intermediate sequence and 3' end sequence of the cyclization element are complementary to the ends of the connection product (belonging to the original linker element A and linker element B), respectively, and are connected under the catalysis of a high-temperature ligase (Taq DNA ligase or ampligase) to form a partially double-stranded circular nucleic acid structure.

[0015] In the present invention, the term "normal temperature" generally refers to the temperature range in which the system is not subjected to a temperature-raising treatment and the cyclization element is in a hairpin structure, for example, 20-37°C; however, as long as the cyclization element is still in the hairpin structure, even if the temperature is not within this range, it is still considered as "normal temperature" in the present invention.

[0016] In some preferred embodiments, in step (iii), a rolling circle amplification reaction is performed starting from the 3' end of the circularization element to obtain the sequencing library.

[0017] The rolling circle amplification reaction can be performed under the action of a displacement enzyme such as phi29 DNA polymerase, Bst DNA polymerase, or Bsm DNA polymerase.

[0018] In some preferred embodiments, in the circularization element of step (ii), the length of the single-end reverse complementary nucleic acid sequence is 5-20 nt; and / or the length of the single-stranded nucleic acid molecule is 15-60 nt.

[0019] Preferably, the 5' ends of the first nucleotide chains of linker element A and linker element B are phosphorylated, and the 3' ends of the first nucleotide chain of linker element A and the second nucleotide chain of linker element B have sticky T ends.

[0020] In some preferred embodiments, the linker element pair further includes one or more of a sequencing primer sequence, a sample tag sequence, and a molecular tag sequence.

[0021] In some more preferred embodiments, the sequencing primer sequence, sample tag sequence, and molecular tag sequence can all be located in one of the adapter elements of the adapter element pair, or can be distributed in both adapter elements. Herein, the sequencing primer sequence, as the name implies, is used for sequencing, the sample tag sequence is used to label samples from different subjects, and the molecular tag sequence is used to label nucleic acid fragments to be tested from the same subject or different subjects.

[0022] In some preferred embodiments, step (i) further comprises performing DNA fragmentation and end-repair reactions on the nucleic acid fragment to be detected.

[0023] Among them, the DNA fragmentation refers to the interruption of sample nucleic acid using physical or chemical methods; preferably, mechanical force or enzymatic cleavage is used to perform the DNA fragmentation; the end repair refers to the end repair of the fragmented DNA molecules to obtain the nucleic acid fragments to be tested.

[0024] The second aspect of the present invention provides a kit for preparing a sequencing library, wherein the kit comprises a linker element pair and a circularization element defined in the preparation method described in the first aspect of the present invention.

[0025] In some preferred embodiments, the kit further comprises one or more of the following components:

[0026] (1) DNA ligase, used to connect the linker element pair and the nucleic acid fragment to be detected.

[0027] (2) Taq DNA ligase, used to connect the 5' and 3' ends of the long chain to form a circular structure.

[0028] (3) A displacement enzyme for rolling circle amplification reaction, preferably phi29 DNA polymerase, Bst DNA polymerase, or Bsm DNA polymerase.

[0029] Preferably, the kit further comprises a reaction buffer comprising 5-500 nM Tris-HCl, 1-500 mM Mg 2+ , 1-500mM K + , 1-50 mM DTT, 1-50 mM ATP, 1-50 mM NAD1, pH 7.0-8.0. Preferably, the 1× working solution of the reaction buffer comprises 25 mM Tris-HCl, 10 mM MgCl2, 25 mM KCl, 10 mM DTT, 1 mM ATP, 1 mM NAD1, pH 7.6.

[0030] A third aspect of the present invention provides a method for preparing a nucleic acid library to be tested in a single tube, wherein the preparation method comprises the following steps:

[0031] (i) providing a nucleic acid fragment to be detected;

[0032] (ii) adding a linker element pair and a cyclization element, and reacting the linker element pair with the nucleic acid fragment to be detected under appropriate conditions to induce ligation and cyclization reaction; wherein the linker element pair comprises a linker element A and a linker element B, wherein both the linker element A and the linker element B are composed of a first nucleotide chain and a second nucleotide chain, and the first nucleotide chain and the second nucleotide chain form a partially complementary double-stranded structure; the cyclization element is a single-stranded nucleic acid molecule having reverse-complementary nucleic acid sequences at both ends, and the middle sequence and 3' end sequence of the cyclization element are complementary to partial sequences of the linker element A and linker element B, respectively;

[0033] (iii) The product of step (ii) is directly subjected to an extension reaction under appropriate conditions without purification or transfer to obtain the library to be tested.

[0034] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0035] The reagents and raw materials used in the present invention are commercially available.

[0036] The beneficial effects brought about by the technical solution of the present invention are:

[0037] 1. The original circularization step in the DNB library preparation process is not required, and the library circularization and DNB preparation are completed during the adapter ligation process;

[0038] 2. The traditional three-step reaction is reduced to a single independent reaction of the present invention, which reduces time, simplifies steps and avoids pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is a schematic diagram of the reaction principle.

[0040] Figure 2 shows the structural sequence and complementary diagram of cyclization element C. DETAILED DESCRIPTION

[0041] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods without specific conditions in the examples were performed according to conventional methods and conditions, or selected according to the product specifications.

[0042] Example 1

[0043] Complete sequencing adapter ligation, product single-strand circularization, and DNB preparation in a single reaction system. Develop a system compatible with TA ligation, single-strand circularization, and rolling circle amplification. The occurrence of different reactions is controlled by temperature.

[0044] Develop a bubble-shaped adapter structure (adapter element) that can be ligated to the target fragment at room temperature (e.g., 20-37°C). The ligated product melts at high temperatures (60-80°C) to form a single-stranded structure that can complement other free single-stranded DNA fragments.

[0045] A single-stranded hairpin structure (cyclization element, i.e., link oligo, see Figures 1 and 2 for examples) is developed. At room temperature (e.g., 20-37°C), the hairpin structure is formed. At high temperatures (above 60°C), the hairpin structure opens and can complement other single-stranded DNA.

[0046] Detailed experimental steps:

[0047] 1. The invention includes a reaction system that is compatible with room-temperature ligase (DNA ligase), high-temperature ligase (Taq DNA ligase) and DNA polymerase with displacement activity (such as BST / Bsm). Through temperature control, different reactions such as linker ligation, DNA circularization and rolling circle replication (also known as rolling circle amplification reaction, used for DNB preparation) can be completed in the same reaction system.

[0048] 2. The buffer of the reaction system contains Tris-HCl (5-500mM), Mg 2+ (preferably MgCl2 1-500mM), K + (KCl, 1-500mM is preferred), DTT (1-50mM), ATP (1-50mM), and NAD1 (1-50mM), with a pH value between 7.0 and 8.0, to ensure that the three enzymes can react normally.

[0049] The preferred buffer is:

[0050] Reagent Name 1× Working Buffer 5× Working Buffer Tris-HCl 25mM 125mMM MgCl 2 10mM 50mMKCl 25mM 125mMDTT 10mM 50mM ATP 1mM 5mMN ADD 11mM 5mMPH 7.67.6

[0051] 3. The reaction system includes linker element A, linker element B and cyclization element C.

[0052] 4. Both linker elements A and B are composed of two partially reverse-complementary nucleotide chains (also referred to as the first nucleotide chain and the second nucleotide chain in the present invention), with a non-complementary DNA sequence in the middle (see Figures 1 and 2); one nucleotide chain of linker element A is phosphorylated at the 5' end and has a 3' sticky T end, and the other nucleotide chain is phosphorylated at the 5' end; one nucleotide chain of linker element B is phosphorylated at the 5' end, and the other nucleotide chain has a 3' sticky T end. The denaturation (melting) temperature (Tm) of the complementary sequence at the connecting end of the linker element and the nucleic acid fragment to be tested is relatively high, which is used to stabilize the double-stranded structure of the linker. The complementary sequence far from the end of the nucleic acid fragment to be tested has a lower Tm and will automatically melt during the subsequent circularization process to form a free end; the linker element carries a sequencing primer sequence, a sample tag sequence, a molecular tag sequence (which can be located in linker element A or linker element B), etc. for downstream sequencing reactions (as shown in Figure 1).

[0053] 5. Circularization element C is a single-stranded nucleic acid molecule with reverse complementarity at its 5' and 3' ends. Its intermediate sequence and 3' end are partially complementary to the DNA sequences in adapter element A and adapter element B, respectively (as shown in Figure 2a and b). Circularization element C forms a hairpin structure (as shown in Figure 2a) at room temperature (typically 20-37°C). It becomes linear at 60-80°C or even higher, enabling it to initiate rolling circle amplification.

[0054] 6. After fragmentation and A addition reaction, double-stranded DNA or natural double-stranded DNA with a phosphorylated 5' end and a sticky end A at the 3' end (collectively referred to as the test nucleic acid fragment) is connected to the adapter element A and the adapter element B under the action of DNA ligase to form a ligation product of adapter element A-test nucleic acid fragment-adapter element B, which is also referred to as a double-stranded DNA structure in this article.

[0055] 7. At 65°C, the terminal DNA double strands of the linker element A-test nucleic acid fragment-linker element B fragment melt and become free single stranded ends, which complementarily pair with the circularization element C to form a double-stranded structure with a local gap, and are then connected into a ring under the action of Taq DNA Ligase.

[0056] 8. Under the action of the replacement enzyme, the rolling circle amplification reaction starts at the 3' end of the circularization element C to obtain the DNB library product for sequencing.

[0057] Table 1 Examples of kit components

[0058]

[0059]

[0060] As shown in Table 1, adapter element A (10 μM) is formed by annealing two oligos, adapter element AF (10 μM) and adapter element AR (10 μM); adapter element B (10 μM) is formed by annealing two oligos, adapter element BF (10 μM) and adapter element BR (10 μM); N in adapter element BR is the sample tag sequence used to mark sample information; the 5' ends of adapter element AF / adapter element AR / adapter element BR are all phosphorylated for subsequent ligation reactions.

[0061] In the linker element, the underlined sequence of one nucleotide chain is complementary to the underlined sequence of the other nucleotide chain, and the bold sequence of one nucleotide chain is complementary to the bold sequence of the other nucleotide chain; in the cyclization element, the underlined sequence at the 5' end of the nucleotide chain is complementary to the underlined sequence at the 3' end.

[0062] Example 2

[0063] Experimental design:

[0064] DNA fragmentation was performed using NA12878 commercial standard DNA. 10 ng of DNA was then used to prepare whole-genome libraries according to the invention method and conventional methods. The libraries were sequenced on a DNBSEQ-2000 sequencer with a sequencing type of PE100 and a sequencing depth of 30×. Data analysis was then performed, including data utilization, alignment rate, duplication rate, GC preference, and other performance. Conventional library construction was performed using the library construction kit (Hieff Biotech Co., Ltd., Shanghai) OnePot II DNA Library Prep Kit for MGI, Catalog No. 13321ES96) was used to prepare the library strictly according to the instructions.

[0065] Inventive method experimental steps:

[0066] 1. DNA fragmentation, end repair and A addition

[0067] DNA was obtained from a commercial standard (NA12878) and was fragmented, end-repaired, and A-tailed using reagents from Shanghai Yisheng Biotechnology Co., Ltd. (kit name: Hieff Fast-Pace DNA Fragmentation Reagent, Catalog No. 12609ES24), as shown in Table 2.

[0068] Table 2 Reaction system for DNA fragmentation, end repair and A addition

[0069]

[0070] The reaction was carried out according to the following procedure:

[0071] 37℃10min72℃20min

[0072] The reaction product was purified using 30 μL XP beads (Agencourt AMPure XP magnetic beads, product number A63881, Beckman Co.), and the obtained product was dissolved in 35 μL TE;

[0073] 2. Ligation and cyclization reactions

[0074] The DNA fragments obtained in the previous step were prepared into a 200 μL centrifuge tube according to the following table (as shown in Table 3). The enzymes were all from NEB (Taq DNA ligase product number M0208S, DNA ligase product number M0202S, and Bst 3.0 product number M0374S).

[0075] Table 3 Reaction systems for ligation and cyclization

[0076] DNA 33μL 5× Working Buffer 5μL DNA Ligase2μL Taq DNA ligase2μLst 3.02μL dNTP (10mM) 2μL Adapter A (0.5μM) 1μL Adapter B (0.5μM) 1μL

[0077] 2 μL of cyclization element C (10 μM) for a total of 50 μL

[0078] The reaction was carried out according to the following procedure:

[0079] 23℃10min65℃25min

[0080] 3. DNB quantification

[0081] The obtained products were quantified by DNB using Quibt ssDNA kit.

[0082] 4. Sequencing

[0083] The qualified DNB prepared in the previous step was sequenced on the sequencing platform DNBSEQ-2000 and the sequencing type PE100.

[0084] 5. Data Analysis

[0085] The analysis included basic steps such as filtering adapter primer sequences and alignment. MGI was assessed using FastQC (version v0.11.8), and PCR duplications were detected using PRINSEQ (version v0.20.4). Raw data were filtered using the NGS QC Toolkit (version v2.3.3). After removing low-quality data and data containing adapters, the remaining data were subjected to the next analysis step: alignment. The filtered data were aligned to the human genome (version GRCh38) using the mem module of BWA (version v0.7.12). Repeats were marked using Picard (version v2.6.0), and local realignment and recalibration of base quality values ​​were performed on the bam files using GATK (version v3.3). SNP and indel variant detection was also performed using GATK, and the resulting variant files were annotated and analyzed with the dbSNP library. Coverage analysis was performed using SAMtools (version v1.9).

[0086] Table 4. Statistics of disembarkation data

[0087] Of clean readsClean read depthMapping rateInventive method931,256,14731.299.63%Conventional method911,245,11230.799.51%Duplicates rateCoverageCoverage of at least 10×Inventive method0.30%99.31%99.72%

[0088] Conventional method 12.10% 99.21% 99.32%

[0089] Table 5. Mutation analysis

[0090] Total SNVsTotal SNVs in dbSNP rateHomozygousHeterozygousInventive method4297973299.77%17280902570597Conventional method4186532199.54%17083922477953Total IndelsTotal Indels in dbSNPdbSNP rate Inventive method79253679098799.80%Conventional method68774268563299.69%

[0091] Results: As shown in the data in Tables 4 and 5, the inventive method can greatly simplify the experimental steps and effectively improve the sequencing performance. The duplication rate (duplicates rate) is much lower than that of conventional methods, and the number of mutations (total SNPs and total indels) detected is also greater than that of conventional methods.

Claims

1. A method for preparing a sequencing library, characterized in that: The preparation method comprises the following steps: (i) obtaining a nucleic acid fragment to be detected; (ii) adding a linker element pair and a cyclization element to undergo ligation and cyclization reactions with the nucleic acid fragment to be detected; wherein the linker element pair comprises a linker element A and a linker element B, wherein both the linker element A and the linker element B are composed of a first nucleotide chain and a second nucleotide chain, and the first nucleotide chain and the second nucleotide chain form a partially complementary double-stranded structure; the cyclization element is a single-stranded nucleic acid molecule having reverse-complementary nucleic acid sequences at both ends, and the middle sequence and 3' end sequence of the cyclization element are complementary to partial sequences of the linker element A and linker element B, respectively; (iii) Perform an extension reaction to obtain a sequencing library.

2. The preparation method according to claim 1, characterized in that In step (ii), the linker element pair is connected to the two ends of the nucleic acid fragment to be tested to form a nucleic acid fragment with different linker elements at both ends. Then, the nucleic acid fragment with different linker elements at both ends is complementary hybridized with the cyclization element to form a partially double-stranded circular nucleic acid structure.

3. The preparation method according to claim 1 or 2, characterized in that In step (iii), a rolling circle amplification reaction is performed starting from the 3' end of the circularization element to obtain the sequencing library.

4. The preparation method according to claim 1, characterized in that In the circularization element of step (ii), the length of the single-end reverse complementary nucleic acid sequence is 5-20 nt; and / or the length of the single-stranded nucleic acid molecule is 15-60 nt; Preferably, the 5' ends of the first nucleotide chains of linker element A and linker element B are phosphorylated, and the 3' ends of the first nucleotide chain of linker element A and the second nucleotide chain of linker element B have sticky T ends.

5. The preparation method according to claim 1, wherein The linker element pair further includes one or more of a sequencing primer sequence, a sample tag sequence, and a molecular tag sequence.

6. The preparation method according to claim 5, wherein The sequencing primer sequence, sample tag sequence, and molecular tag sequence may all be located in one of the linker elements of the linker element pair, or may be distributed in two linker elements.

7. The preparation method according to claim 1, wherein In step (i), the method further comprises performing DNA fragmentation and end-repair reaction on the nucleic acid fragment to be detected.

8. A kit for preparing a sequencing library, characterized in that: The kit comprises the linker element pair and the cyclization element defined in the preparation method according to any one of claims 1 to 7.

9. The kit according to claim 8, wherein The kit may further comprise one or more of the following components: (1) DNA ligase, used to connect the linker element pair and the nucleic acid fragment to be detected; (2) Taq DNA ligase, used to connect the 5' and 3' ends of the long chain to form a circular structure; (3) A displacement enzyme for rolling circle amplification reaction, preferably phi29 DNA polymerase, Bst DNA polymerase, or Bsm DNA polymerase.

10. A method for preparing a nucleic acid library to be tested in a single tube, characterized in that: The method comprises the following steps: (i) providing a nucleic acid fragment to be detected; (ii) adding a linker element pair and a cyclization element as defined in any one of claims 1 to 7, and causing ligation and cyclization reactions with the nucleic acid fragment to be tested under appropriate conditions; wherein the linker element pair comprises a linker element A and a linker element B, each of the linker element A and the linker element B being composed of a first nucleotide chain and a second nucleotide chain, the first nucleotide chain and the second nucleotide chain forming a partially complementary double-stranded structure; the cyclization element is a single-stranded nucleic acid molecule having reverse-complementary nucleic acid sequences at both ends, and the middle sequence and 3' end sequence of the cyclization element are complementary to partial sequences of the linker element A and linker element B, respectively; (iii) The product of step (ii) is directly subjected to an extension reaction under appropriate conditions without purification or transfer to obtain the library to be tested.

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