Construction and application of DNA library

Abstract

The present invention provides a method for constructing a DNA library and its application. The method for constructing the DNA library includes: a) connecting a first connector and a second connector at both ends of a sample DNA to obtain a connector-connected DNA; b) using a primer that can be complementary paired with the first connector as a single primer, performing unidirectional PCR amplification on the connector-connected DNA to obtain a unidirectional PCR amplification library; c) using a capture probe that can be complementary paired with the second connector to capture the unidirectional PCR amplification library to obtain a capture library-probe complex; d) removing the capture probe from the capture library-probe complex to obtain a DNA library. The method can solve the problem of incomplete fragments in the DNA library in the prior art and is suitable for the field of DNA library construction.

Description

Technical Field

[0001] The present invention relates to the field of DNA library construction, and in particular to a DNA library construction method and application. Background Art

[0002] In the current field of molecular biology research and genomics, DNA library construction is a key technology that provides the necessary material basis for subsequent capture, sequencing and analysis. However, although this technology has made significant progress, in practical applications, researchers face a common problem that the tags at both ends of the sequence in the DNA library may be incomplete.

[0003] Incomplete tags at both ends of the DNA library may involve partial deletion of primers or adapter sequences connected to the DNA fragments. This incompleteness may be caused by various factors during library preparation, including experimental operations, primer design or other technical challenges. The existence of this problem may have serious effects on subsequent experiments, including but not limited to reduced capture efficiency, reduced sequencing quality, and threats to the reliability of data analysis.

[0004] Taking hybridization capture technology as an example, hybridization capture technology is a targeted enrichment technology widely used in high-throughput sequencing. During the process of hybridization with the probe, the sequencing library (hybridization pre-library) is complementary and bound according to sequence similarity, and then the probe and the bound library are captured by the modification group on the probe. In practical applications, generally speaking, the library molecules in the pre-library need to be complete, that is, both ends need to have specific linker sequences for PCR amplification after capture. Incomplete library molecules will be lost after being captured because they cannot be amplified, resulting in loss of genetic information. Summary of the invention

[0005] The main purpose of the present invention is to provide a method for constructing a DNA library and its application, so as to solve the problem of incomplete fragments in the DNA library in the prior art.

[0006] In order to achieve the above-mentioned purpose, according to the first aspect of the present invention, a method for constructing a DNA library is provided, and the construction method comprises: a) connecting a first connector and a second connector at both ends of a sample DNA to obtain connector-connected DNA; b) using a primer that can complementarily pair with the first connector as a single primer, performing one-way PCR amplification on the connector-connected DNA to obtain a one-way PCR amplification library; c) using a capture probe that can complementarily pair with the second connector to capture the one-way PCR amplification library to obtain a capture library-probe complex; d) removing the capture probe in the capture library-probe complex to obtain a DNA library.

[0007] Furthermore, using a)-c) in the construction method, different sample DNAs are processed separately to obtain a capture library-probe complex for each sample DNA; the capture library-probe complex for each sample DNA is mixed to obtain a mixed sample capture library-probe complex; the capture probe in the mixed sample capture library-probe complex is removed to obtain a mixed sample library; wherein, in the construction method for different sample DNAs, the ratio of the molar content of different DNA libraries in the mixed sample library is the same as the ratio of the molar amount of the capture probe added to the DNA library.

[0008] Furthermore, in the construction method of different sample DNAs, the added molar amount of the capture probe is the same; accordingly, the molar content of different DNA libraries in the mixed sample library is the same.

[0009] Furthermore, the capture probe is a single-stranded DNA, and the capture probe carries a modification group for capture; preferably, the modification group is a biotin group.

[0010] Furthermore, magnetic beads capable of capturing the capture probe are added to the capture library-probe complex; preferably, the magnetic beads include streptavidin magnetic beads.

[0011] Furthermore, the removal of the capture probe in the capture library-probe complex includes: adjusting the pH to ≥ 9 to cause DNA denaturation in the capture library-probe complex, removing the magnetic beads to remove the free capture probe; or using a primer that can complementarily pair with the second connector as a primer, performing PCR amplification on the connector-connected DNA, and removing the magnetic beads to remove the free capture probe.

[0012] Furthermore, the capture probe contains uracil. After removing the magnetic beads, the pH is adjusted to 6-8.5, and uracil-DNA glycosylase and uracil-specific excision reagent are added to the system to remove the residual capture probe fragments; preferably, the uracil-specific excision reagent is USER enzyme.

[0013] Furthermore, the mixed sample library is a hybridization pre-library or a library to be sequenced.

[0014] In order to achieve the above object, according to the second aspect of the present invention, there is provided an application of the above DNA library construction method in DNA library construction.

[0015] Furthermore, the application includes application in the construction of hybridization pre-library or high-throughput sequencing library.

[0016] By applying the technical solution of the present invention, in the above-mentioned method for constructing a DNA library, one-way PCR amplification is performed using primers that can specifically bind to the connector on one side, and then the fragments are captured using capture probes that can specifically bind to the connector on the other side, so that a DNA library with a complete sequence can be obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0018] Figure 1 A schematic diagram showing library molecules in different states after amplification by primers at one end and capture by probes at the other end according to an embodiment of the present invention is shown.

[0019] Figure 2 The fragment length distribution results of six FFPE libraries according to Example 1 of the present invention are shown.

[0020] Figure 3 The figure shows the sequencing data volume result after mixed hybridization capture of six FFPE libraries according to Example 1 of the present invention.

[0021] Figure 4 The on-target rate and duplication rate result graph of mixed hybridization capture sequencing of six FFPE libraries according to Example 1 of the present invention is shown. DETAILED DESCRIPTION

[0022] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0023] As mentioned in the background art, in the prior art, when constructing a DNA library, the incomplete fragments present in the library will cause the loss of genetic information in the subsequent capture or sequencing stage. Therefore, in this application, the inventor attempts to develop a new method for constructing a DNA library and proposes a series of protection schemes of this application.

[0024] In a first typical embodiment of the present application, a method for constructing a DNA library is provided, the method comprising: a) connecting a first connector and a second connector at both ends of a sample DNA to obtain connector-connected DNA; b) using a primer that can complementarily pair with the first connector as a single primer, performing unidirectional PCR amplification on the connector-connected DNA to obtain a unidirectional PCR amplification library; c) using a capture probe that can complementarily pair with the second connector to capture the unidirectional PCR amplification library to obtain a capture library-probe complex; d) removing the capture probe from the capture library-probe complex to obtain a DNA library.

[0025] The DNA library in the present application is a collection of DNA fragments, including but not limited to DNA from various sources, such as a DNA library derived from genomic DNA, or a DNA library obtained by reverse transcription of RNA.

[0026] In the above-mentioned method for constructing a DNA library, after connecting the adapters at the 5' end and the 3' end of the sample DNA using the existing technology, a primer that can be complementary paired with the adapter on one side (the adapter connected to the 5' end or the adapter connected to the 3' end) is used as a single primer to perform unidirectional PCR amplification to obtain a unidirectional PCR amplification library. Then, a capture probe that can be complementary paired with the adapter on the other side (the adapter connected to the 3' end or the adapter connected to the 5' end) is used to capture the unidirectional PCR amplification library to obtain a capture library-probe complex. This capture library-probe complex must contain a complete first adapter and a second adapter as well as a DNA fragment in the middle of the adapter. Finally, the capture probe in the capture library-probe complex is removed to obtain a DNA library, which can be used for subsequent on-machine data and can also be further hybridized and captured.

[0027] The schematic diagram of the above construction method is as follows: Figure 1 As shown in the figure, "X" indicates that the DNA fragment cannot be obtained. Among them, a is the correct library molecule and can be captured; b has two ends that are not amplification ends and cannot be amplified; c has two ends that are amplification ends and can be amplified, but cannot be captured; d has a missing adapter sequence at the amplification end and cannot be amplified; e has a missing adapter sequence at the capture end and can be amplified, but cannot be captured.

[0028] In a preferred embodiment, different sample DNAs are processed separately using a)-c) in the construction method to obtain a capture library-probe complex for each sample DNA; the capture library-probe complex for each sample DNA is mixed to obtain a mixed sample capture library-probe complex; the capture probe in the mixed sample capture library-probe complex is removed to obtain a mixed sample library; wherein, in the construction method for different sample DNAs, the molar content ratio of different DNA libraries in the mixed sample library is the same as the molar content ratio of the capture probe added to the DNA library.

[0029] Mixed hybridization of multiple pre-libraries is an important mechanism to reduce costs and experimental operations. In practical applications, in order to achieve mixed hybridization of multiple pre-libraries and produce the expected amount of captured data, each pre-library needs to determine the molar amount (calculate the added volume based on the molar concentration) and then mix as needed. For example, if the mixed sample library contains DNA library A, DNA library B, and DNA library C, in the construction of the DNA library, the molar amounts of probes added to DNA libraries A, B, and C are A1, B1, and C1, respectively, and the molar amounts of DNA libraries A, B, and C in the mixed sample library are A2, B2, and C2, respectively, then A1:B1:C1=A2:B2:C2.

[0030] Using the above construction method, when capturing the sample DNA, the molar amount of the capture probe is theoretically the same as the molar amount of the DNA library obtained subsequently, and the difference is also very small in practical applications. Therefore, by adjusting the molar amount of the added capture probe, the amount of the DNA library can be controlled, and the amount of each DNA library in the mixed sample of multiple DNA samples can be further controlled. There is no need to measure the concentration and calculate the added volume after obtaining the DNA library separately. Using the above construction method, the control of the content of multiple DNA libraries (including absolute content (actual molar amount in the system) and relative content (proportion in the mixed system)) is achieved only by controlling the amount of capture probe added. This construction method is easy to implement in experimental automation, thereby improving the construction efficiency of libraries, especially hybrid capture pre-libraries.

[0031] In a preferred embodiment, in the construction method of different sample DNAs, the added molar amount of the capture probe is the same; accordingly, the molar contents of different DNA libraries in the mixed sample library are the same.

[0032] In the above construction method, if the molar amount of the capture probe added to different sample DNAs is the same, the molar amount of the capture library-probe complex of each sample DNA is the same, and the molar amount of each DNA library in the mixed system after removing the capture probe is also the same.

[0033] In a preferred embodiment, the capture probe is a single-stranded DNA, and the capture probe carries a modification group for capture; preferably, the modification group is a biotin group.

[0034] In a preferred embodiment, magnetic beads capable of capturing the capture probe are added to the capture library-probe complex; preferably, the magnetic beads include streptavidin magnetic beads.

[0035] The capture probe may optionally be a single-stranded DNA containing a modification group. Such a modification group can be used to efficiently separate the capture library-probe complex from the system, including but not limited to using magnetic beads.

[0036] In a preferred embodiment, the removal of the capture probe in the capture library-probe complex includes: adjusting the pH ≥ 9 (including but not limited to pH = 9-14, 9-13, 9-12, 9-11 or 9-10) to cause DNA denaturation in the capture library-probe complex, removing the magnetic beads to remove the free capture probe; or using a primer that can complementarily pair with the second connector as a primer, performing PCR amplification on the connector-connected DNA, and removing the magnetic beads to remove the free capture probe.

[0037] In a preferred embodiment, the capture probe contains uracil. After removing the magnetic beads, the pH is adjusted to 6-8.5, and uracil-DNA glycosylase and uracil-specific excision reagent are added to the system to remove the residual capture probe fragments; preferably, the uracil-specific excision reagent is USER enzyme.

[0038] The uracil-specific excision reagent is an enzyme that can cleave DNA at the position of uracil.

[0039] In a preferred embodiment, the mixed sample library is a hybridization pre-library or a library to be sequenced.

[0040] In a second typical embodiment of the present application, an application of the above-mentioned library construction method in DNA library construction is provided.

[0041] In a preferred embodiment, the application includes application in the construction of a hybridization pre-library or a high-throughput sequencing library.

[0042] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0043] Example 1

[0044] Preparation of hybridization pre-libraries for formaldehyde-fixed paraffin-embedded (FFPE) samples.

[0045] Due to the accumulation of DNA damage in FFPE samples, the library preparation efficiency is low, and there are also large differences in the libraries prepared from FFPE samples in different states. Through conventional quality inspection processes, it is often impossible to accurately quantify the molar amount of effective library molecules in the library, which results in uneven data yields from mixed hybridization.

[0046] Six DNA libraries of FFPE samples were selected for Illumina sequencing platform. The fragment length distribution of the six libraries is as follows Figure 2 As shown. According to the fragment length and The mass concentration was measured by 3.0 fluorescence quantitative instrument (Thermo Fisher), and six libraries were mixed according to Table 1 as a control group.

[0047] Table 1 Quality inspection of six FFPE libraries

[0048] Mass concentration (ng / μL) Average length (bp) Control group mixed volume (μL) Library 1 34.6 245 14.5 Library 2 44.1 316 14.6 Library 3 40.9 318 15.9 Library 4 57.3 327 11.6 Library 5 56.0 335 12.2 Library 6 30.2 336 22.7

[0049] According to the method provided by the present invention, 10 μL of each of the six libraries was taken and added to the reaction system shown in Table 2, and amplified on a PCR instrument. The amplification cycle was 95 degrees for 15 seconds, 55 degrees for 15 seconds, and 72 degrees for 1 minute, and the number of cycles was 10.

[0050] Table 2 Library single-end primer amplification system

[0051] Components Volume (μL) KAPA HiFi HotStart ReadyMix (Roche) 12.5 P5 primer (10pmol / μL) 2.5

[0052] The sequence of the P5 primer is: AATGATACGGCGACCACC (SEQ ID NO: 1).

[0053] P7U probe was added to the amplified systems of the six libraries at a concentration of 100 fmol / μL in a volume of 4 μL and incubated at 60 degrees for 15 minutes.

[0054] The sequence of the P7U probe is 5'biotin-AUAGCAGAAGACGGCAUACGAGAAAAAAAAAGTGACUGGAGTTCAGACGUGTGCTCTGA (SEQ ID NO: 2), wherein positions 2, 17, 37 and 50 are uracil (U) and the other positions are DNA bases (A, T, C and G).

[0055] The six systems were mixed at room temperature, added to 50 μL of washed streptavidin magnetic beads (Thermo Fisher, M270), and incubated at 60 degrees for 30 minutes.

[0056] Collect the magnetic beads on a magnetic stand, wash once with wash buffer (1xSSPE, 0.1% Triton x-100), collect the magnetic beads again, and discard the supernatant. Add the reaction system shown in Table 3, and amplify on a PCR instrument. The amplification cycle is 95 degrees for 15 seconds, 55 degrees for 15 seconds, and 72 degrees for 1 minute. The number of cycles is 2. Then incubate at 65 degrees for 15 minutes.

[0057] Table 3 Prelibrary second strand generation amplification system

[0058] Components Volume (μL) KAPA HiFi HotStart ReadyMix(Roche) 12.5 P7 primer (10 pmol / μL) 2.5 Thermostable USER III Enzyme(NEB) 1 Nuclease-free water 9

[0059] Collect the supernatant on a magnetic stand and add 1x DNA purification magnetic beads ( SP Beads) to obtain a mixed hybridization pre-library.

[0060] The control group and the hybridization pre-library prepared according to this method were subjected to whole exome capture sequencing using NEXome Core Panel (Naonda). Figure 3 As shown. The data volume of the control group was quite different, which was not in line with expectations, and additional sequencing was required to obtain sufficient data volume. Using this method, basically consistent data volume was obtained, and no additional sequencing was required. Figure 4 As shown in the figure, the hybridization capture target rate is basically the same as that of the control group. The repetitiveness of sequencing data has not been significantly improved, which is because the additional amplification step is not exponential amplification.

[0061] Example 2

[0062] Preparation of plasma free DNA hybridization prelibrary.

[0063] Two plasma free DNA (cfDNA) Illumina platform paired-end molecular tag sequencing libraries, libA was constructed with a starting amount of 10ng cfDNA, and libB was constructed with a starting amount of 50ng cfDNA.

[0064] According to the method provided by the present invention, 20 μL of libA and libB were respectively added to the reaction system with the same proportion as in Table 2, and amplified on a PCR instrument. The amplification cycle was 95 degrees for 15 seconds, 55 degrees for 15 seconds, and 72 degrees for 1 minute, and the number of cycles was 10.

[0065] After amplification, add 100 fmol / μL of P7U probe (SEQ ID NO: 2) to the system, 1 μL to libA product and 5 μL to libB product, and incubate at 60 degrees for 15 minutes.

[0066] The above system was mixed at room temperature, added to 50 μL of washed streptavidin magnetic beads (Thermo Fisher, M270), and incubated at 60 degrees for 30 minutes.

[0067] The magnetic beads were collected on a magnetic stand, washed once with wash buffer (1xSSPE, 0.1% Triton x-100), the magnetic beads were collected again, and the supernatant was discarded.

[0068] Add 15 μL of freshly prepared 0.1 N NaOH solution to the magnetic beads, mix and let stand at room temperature for 5 minutes, add 3 μL of 1 M Tris-HCl pH 7.4, mix and collect the supernatant on a magnetic stand.

[0069] Use of supernatant HotSpot Panel v1.0 (Naonda) probe and Hybrid Capture Reagents (Naonda) were used for hybrid capture.

[0070] As shown in Table 4, the ratio of the sequencing data volume of libA and libB is basically consistent with the added volume ratio of the P7U probe. The repetition rate of the two data in the target area is similar, which avoids over-sequencing of samples with low starting input and under-saturation of samples with high starting input.

[0071] Table 4. Actual sequencing results of hybridization pre-library prepared at 1:5

[0072] Expected data volume (Gb) Actual data volume (Gb) Average coverage depth Target area repetition rate LibA 1 1.2 14555 92.1％ LibB 5 5.3 63261 92.5％

[0073] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: by using the above DNA library construction method, using one-way PCR amplification and specific probe capture, a DNA library with complete sequence can be obtained. By adding a specific amount of specific probes, the control of the DNA fragment content in the library can be achieved. When mixing DNA libraries from different sources later, there is no need to quantify each pre-library separately, calculate the mixing volume, and mix them one by one, which is more conducive to automation.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for constructing a DNA library, characterized in that: The construction method comprises: a) connecting a first adapter and a second adapter at both ends of the sample DNA to obtain adapter-connected DNA; b) using a primer that can complementarily pair with the first adapter as a single primer, performing unidirectional PCR amplification on the adapter-connected DNA to obtain a unidirectional PCR amplification library; c) capturing the one-way PCR amplification library using a capture probe that can complementarily pair with the second linker to obtain a capture library-probe complex; d) removing the capture probe from the capture library-probe complex to obtain the DNA library.

2. The construction method according to claim 1, characterized in that: Using a) to c) in the construction method, respectively process different sample DNAs to obtain a capture library-probe complex of each sample DNA; Mixing the capture library-probe complex of each sample DNA to obtain a mixed sample capture library-probe complex; removing the capture probe from the mixed sample capture library-probe complex to obtain a mixed sample library; Wherein, in the construction method for different sample DNAs, the ratio of the molar contents of different DNA libraries in the mixed sample library is the same as the ratio of the molar amounts of the capture probes added to the DNA library.

3. The construction method according to claim 2, characterized in that: In the construction methods of different sample DNAs, the added molar amounts of the capture probes are the same; accordingly, the molar contents of different DNA libraries in the mixed sample library are the same.

4. The construction method according to claim 1, characterized in that: The capture probe is a single-stranded DNA, and a modification group for capture is carried on the capture probe.

5. The construction method according to claim 4, characterized in that: The modifying group is a biotin group.

6. The construction method according to claim 4 or 5, characterized in that: Magnetic beads capable of capturing the capture probes are added to the capture library-probe complex.

7. The construction method according to claim 6, characterized in that: The magnetic beads include streptavidin magnetic beads.

8. The construction method according to claim 6, characterized in that: The removal of the capture probe in the capture library-probe complex comprises: Adjusting the pH to ≥ 9 to allow the capture library-probe complex to undergo DNA denaturation, and removing the magnetic beads to remove the free capture probes; or The primers capable of complementary pairing with the second linker are used as primers to perform PCR amplification on the linker-connected DNA, and the magnetic beads are removed to remove the free capture probes.

9. The construction method according to claim 8, characterized in that: The capture probe contains uracil. After removing the magnetic beads, the pH is adjusted to 6-8.5, and uracil-DNA glycosylase and uracil-specific excision reagent are added to the system to remove the residual fragments of the capture probe.

10. The construction method according to claim 9, characterized in that: The uracil-specific excision reagent is USER enzyme.

11. The construction method according to claim 2 or 3, characterized in that: The mixed sample library is a hybridization pre-library or a library to be sequenced.

12. Use of the library construction method according to any one of claims 1 to 11 in the construction of a DNA library.

13. The use according to claim 12, characterized in that: The application includes application in the construction of hybridization pre-library or high-throughput sequencing library.

Images