CN118086457B

CN118086457B - Construction and application of DNA library

Info

Publication number: CN118086457B
Application number: CN202410199044.5A
Authority: CN
Inventors: 汪彪; 曲燕; 吴强
Original assignee: Naonda Nanjing Biological Technology Co ltd
Current assignee: Naonda Nanjing Biological Technology Co ltd
Priority date: 2024-02-22
Filing date: 2024-02-22
Publication date: 2025-03-11
Anticipated expiration: 2044-02-22
Also published as: CN118086457A

Abstract

The present invention provides a method for constructing a DNA library and its application. The method for constructing the above-mentioned 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

Construction method and application of DNA library

Technical Field

The invention relates to the field of DNA library construction, in particular to a construction method and application of a DNA library.

Background

In the current fields 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 technique has made remarkable progress, in practical use, researchers are faced with a ubiquitous problem in that the tags at both ends of the sequence in a DNA library may be incomplete.

Incomplete labeling of both ends of a DNA library may involve partial deletion of primer or adapter sequences attached to the DNA fragments. Such imperfections may result from various factors in the library preparation process, including experimental manipulations, primer design, or other technical challenges. The existence of this problem can have serious impact on subsequent experiments including, but not limited to, reduced capture efficiency, reduced sequencing quality, and compromised reliability of data analysis.

Taking the hybrid capture technique as an example, the hybrid capture technique is a targeted enrichment technique that is widely used in high throughput sequencing. In the process of hybridization with the probe, the sequencing library (hybridization pre-library) is complementarily combined according to sequence similarity, and then the probe and the combined library are captured through the modification group on the probe. In practice, the library molecules in the pre-library typically need to be intact, i.e., they need to bear specific linker sequences at both ends, for post-capture PCR amplification. Incomplete library molecules are lost after being captured due to inability to amplify, resulting in loss of genetic information.

Disclosure of Invention

The invention mainly aims to provide a construction method and application of a DNA library, which are used for solving the problem that incomplete fragments exist in the DNA library in the prior art.

In order to achieve the above object, according to a first aspect of the present invention, there is provided a method for constructing a DNA library, comprising a) ligating a first adaptor and a second adaptor at both ends of a sample DNA to obtain adaptor-ligated DNA, b) performing one-way PCR amplification on the adaptor-ligated DNA with a primer capable of complementarily pairing with the first adaptor as a single primer to obtain a one-way PCR amplified library, c) capturing the one-way PCR amplified library with a capture probe capable of complementarily pairing with the second adaptor to obtain a capture library-probe complex, and d) removing the capture probe in the capture library-probe complex to obtain a DNA library.

Further, using a) -c) in the construction method, respectively processing different sample DNAs to obtain a capture library-probe complex of each sample DNA, mixing the capture library-probe complexes of each sample DNA to obtain a mixed sample capture library-probe complex, and removing the capture probes in the mixed sample capture library-probe complex to obtain a mixed sample library, wherein in the construction method for the different sample DNAs, the ratio of the molar contents of the different DNA libraries in the mixed sample library is the same as the ratio of the molar amounts of the capture probes added in the DNA library.

Further, in the construction method of different sample DNAs, the molar quantity of the capture probes added is the same, and correspondingly, the molar contents of different DNA libraries in the mixed sample library are the same.

Further, the capture probe is a single-stranded DNA, and the capture probe is provided with a modification group for capturing, preferably a biotin group.

Further, magnetic beads capable of capturing capture probes, preferably comprising streptavidin magnetic beads, are added to the capture library-probe complexes.

Further, the removal of the capture probes in the capture library-probe complex comprises the steps of adjusting the pH to be more than or equal to 9 so that DNA melting occurs in the capture library-probe complex, removing magnetic beads to remove free capture probes, or carrying out PCR amplification on the adaptor-ligated DNA by taking a primer capable of complementarily pairing with the second adaptor as a primer, and removing the magnetic beads to remove the free capture probes.

Further, the capture probe contains uracil, after removing the magnetic beads, the pH is adjusted to 6-8.5, uracil-DNA glycosylase and uracil specific excision reagent are added into the system, and the fragments of the residual capture probe are removed, preferably the uracil specific excision reagent is USER enzyme.

Further, the mixed sample library is a hybridization pre-library or a library to be sequenced.

In order to achieve the above object, according to a second aspect of the present invention, there is provided the use of the above method for constructing a DNA library in the construction of a DNA library.

Further, applications include use in the construction of hybridization pre-libraries or high throughput sequencing libraries.

By applying the technical scheme of the invention, in the construction method of the DNA library, the primer capable of being specifically combined with one side joint is utilized to carry out unidirectional PCR amplification, and then the capture probe capable of being specifically combined with the other side joint is utilized to capture fragments, so that the DNA library with complete sequence can be obtained.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:

FIG. 1 is a schematic diagram showing the condition of library molecules in different states after amplification of one end primer and capture of the other end probe according to an embodiment of the present invention.

FIG. 2 shows a graph of the distribution of the lengths of six FFPE library fragments in accordance with example 1 of the present invention.

FIG. 3 shows a plot of sequencing data volume results after hybrid hybridization capture for six FFPE libraries according to example 1 of the present invention.

FIG. 4 shows a plot of mid-target and repetition rate results of hybrid capture sequencing of six FFPE libraries according to example 1 of the present invention.

Detailed Description

It should be noted that, without conflict, the embodiments of the present application and features of the embodiments may be combined with each other. The present application will be described in detail with reference to examples.

As mentioned in the background, in the prior art, when constructing DNA libraries, incomplete fragments present in the library may cause loss of genetic information during subsequent capture or sequencing phases. Thus, in the present application, the inventors have tried to develop a new construction method of DNA library, and proposed a series of protection schemes of the present application.

In a first exemplary embodiment of the present application, there is provided a method for constructing a DNA library, comprising a) ligating a first adaptor and a second adaptor at both ends of a sample DNA to obtain adaptor-ligated DNA, b) performing one-way PCR amplification on the adaptor-ligated DNA with a primer capable of complementarily pairing with the first adaptor as a single primer to obtain a one-way PCR amplified library, c) capturing the one-way PCR amplified library with a capture probe capable of complementarily pairing with the second adaptor to obtain a capture library-probe complex, and d) removing the capture probe in the capture library-probe complex to obtain a DNA library.

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

In the method for constructing a DNA library, a primer capable of complementary pairing with one side of a linker (a linker attached to the 5 'end or a linker attached to the 3' end) is used as a single primer after the linkers are attached to the 5 'end and the 3' end of a sample DNA by using the prior art, and unidirectional PCR amplification is performed to obtain a unidirectional PCR amplified library. And capturing the unidirectional PCR amplified library by using a capturing probe which can be complementarily paired with a connector at the other side (a connector connected at the 3 'end or a connector connected at the 5' end) to obtain a capturing library-probe complex. Such capture library-probe complexes necessarily contain intact first and second adaptors and DNA fragments intermediate the adaptors. Finally, the capture probes in the capture library-probe complex are removed to obtain a DNA library, and the DNA library can be used for subsequent on-machine data and can also be used for further hybridization capture.

For the case where the adaptor-ligated DNA in different states is amplified by one primer and captured by the other probe. A schematic diagram of the above construction method is shown in FIG. 1, and "X" indicates that a DNA fragment cannot be obtained. Wherein a is a correct library molecule which can be captured, b is not an amplification end, and cannot be amplified, c is an amplification end, and can be amplified, but cannot be captured, d is an amplification end deletion linker sequence, and e is an amplification end deletion linker sequence, and cannot be captured.

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

Multiple pre-library hybrid hybridization is an important mechanism to reduce cost and reduce experimental manipulations. In practice, to achieve mixed hybridization of a plurality of pre-libraries and to yield a volume of captured data that meets the expectations, each pre-library needs to be mixed as desired after determining the molar amount (added volume calculated from molar concentration). If the mixed sample library contains a DNA library A, DNA library B and a DNA library C, in the construction of the DNA library, the molar amounts of the probes added to the DNA libraries A, B and C are A1, B1 and C1, respectively, and the molar amounts of the DNA libraries A, B and C in the mixed sample library are A2, B2 and C2, respectively, then A1:B1:C1=A2:B2:C2.

By using the construction method, the molar quantity of the capture probe is theoretically the same as that of the DNA library obtained later when capturing the sample DNA, and the difference is small in practical application. Thus, by adjusting the molar amount of capture probes added, control over the amount of DNA library can be achieved, further control over the amount of each DNA library in a mixed sample of multiple DNA samples. The concentration need not be determined and the addition volume calculated after the DNA library is obtained separately. With the above construction method, control of the content of various DNA libraries (including absolute content (actual molar amount in the system) and relative content (duty ratio in the mixed system)) is achieved by controlling only the addition amount of the capture probe. The construction method is convenient to realize in experimental automation, thereby improving the construction efficiency of libraries, especially hybridization capture pre-libraries.

In a preferred embodiment, the molar amount of capture probes added is the same in the construction of different sample DNA and accordingly the molar content of different DNA libraries in the mixed sample library is the same.

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

In a preferred embodiment, the capture probe is a single-stranded DNA with a modifying group for capture, preferably a biotin group.

In a preferred embodiment, magnetic beads capable of capturing capture probes, preferably comprising streptavidin magnetic beads, are added to the capture library-probe complexes.

The capture probes are optionally single-stranded DNA containing modification groups that allow efficient separation of capture library-probe complexes from the system, including but not limited to the use of magnetic beads.

In a preferred embodiment, removal of capture probes from the capture library-probe complex comprises adjusting the pH to > 9 (including but not limited to pH=9-14, 9-13, 9-12, 9-11 or 9-10) to cause DNA melting of the capture library-probe complex, removing magnetic beads to remove free capture probes, or PCR amplification of adaptor-ligated DNA using primers capable of complementary pairing to the second adaptor to remove free capture probes.

In a preferred embodiment, the capture probe contains uracil and after removal of the magnetic beads, the pH is adjusted to 6-8.5, uracil-DNA glycosylase and uracil specific excision reagent, preferably the uracil specific excision reagent is a USER enzyme, are added to the system to remove the fragment of the capture probe that remains.

The uracil-specific excision reagent is an enzyme capable of cleaving DNA at the uracil position.

In a preferred embodiment, the pooled sample library is a hybridization pre-library or a library of test sequences.

In a second exemplary embodiment of the present application, there is provided the use of a method of constructing a library as described above in the construction of a DNA library.

In a preferred embodiment, the use comprises use in the construction of a hybridization pre-library or a high throughput sequencing library.

The advantageous effects of the present application will be explained in further detail below in connection with specific examples.

Example 1

Preparation of a formaldehyde-fixed paraffin-embedded (FFPE) sample hybridization pre-library.

FFPE samples have lower library preparation efficiency due to accumulation of DNA damage, and libraries prepared from FFPE samples in different states also have larger differences. Through conventional quality control procedures, the effective library molecular weight in the library often cannot be accurately quantified, and thus the data yield of the hybrid hybridization is uneven.

A DNA library of six FFPE sample Illumina sequencing platforms was selected. The fragment length distribution of the six libraries is shown in FIG. 2. According to the segment length and the segment length3.0 Mass concentrations measured by fluorescence quantitative instrument (Siemens), six libraries were mixed as in Table 1 and used as control.

TABLE 1 six FFPE library quality control cases

	Concentration by mass (ng/. Mu.L)	Average length (bp)	Control mix 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

According to the method provided by the invention, 10 mu L of each of the six libraries was taken, added to the reaction system shown in Table 2, amplified on a PCR apparatus, and the amplification cycle was 95 degrees 15 seconds, 55 degrees 15 seconds, 72 degrees 1 minute, and the number of cycles was 10.

TABLE 2 Single-ended primer amplification System for library

Component (A)	Volume (mu L)
		KAPA HiFi HotStart ReadyMix(Roche)	12.5
P5 primer (10 pmol/. Mu.L)	2.5

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

P7U probe was added to each of the six amplified systems at a concentration of 100 fmol/. Mu.L, and incubated at 60℃for 15 minutes in a volume of 4. Mu.L.

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

All six above systems were mixed at room temperature, added to 50. Mu.L of streptavidin magnetic beads (Sieimer, M270) after washing, and incubated at 60℃for 30 minutes.

The beads were collected on a magnetic rack, washed once with a wash buffer (1 XSSPE,0.1% Triton x-100), collected again, and the supernatant removed. The reaction system shown in Table 3 was added, and amplification was performed on a PCR apparatus with cycles of 95℃for 15 seconds, 55℃for 15 seconds, 72℃for 1 minute, and 2 cycles. Then incubated at 65℃for 15 minutes.

TABLE 3 Pre-library two-strand generation amplification System

Component (A)	Volume (mu L)
		KAPA HiFi HotStart ReadyMix(Roche)	12.5
P7 primer(10pmol/μL)	2.5
		Thermostable USER III Enzyme(NEB)	1
Nuclease-free water	9

Collecting supernatant on a magnetic rack, adding 1x DNA purification magnetic beads into the supernatantSP loads) to obtain a mixed hybridization pre-library.

Control and hybridization pre-library prepared according to the method were sequenced using NEXome Core Panel (Naonda) total exon trap, respectively. The data amount comparison is shown in fig. 3. The control group data amount was widely different from the expected one, and additional sequencing was required to obtain sufficient data amount. A substantially consistent amount of data is obtained using the present method, without the need for additional sequencing. As shown in FIG. 4, the target rate in hybridization capture was substantially consistent compared to the control group. The repetition rate of the sequencing data is not significantly improved, since the additional amplification step added is not an exponential amplification.

Example 2

Preparation of plasma free DNA hybridization Pre-library.

Two illumina platform double-ended molecular tag sequencing libraries of plasma free DNA (cfDNA), libA was pooled for 10ng cfDNA starting amount, and libB was pooled for 50ng cfDNA starting amount.

According to the method provided by the invention, libA and libB were added to a reaction system having the same ratio as in Table 2, and amplification was performed on a PCR apparatus with cycles of 95 degrees 15 seconds, 55 degrees 15 seconds, 72 degrees 1minute, and 10 cycles.

To the amplified system was added a P7U probe (SEQ ID NO: 2) at a concentration of 100 fmol/. Mu.L, respectively, wherein 1. Mu.L was added to libA products and 5. Mu.L was added to libB products. Incubate at 60℃for 15 min.

The above systems were mixed at room temperature, and added to 50. Mu.L of streptavidin magnetic beads (Sieimer, M270) after washing, and incubated at 60℃for 30 minutes.

The beads were collected on a magnetic rack, washed once with a wash buffer (1 XSSPE,0.1% Triton x-100), collected again, and the supernatant removed.

To the beads, 15. Mu.L of freshly prepared 0.1N NaOH solution was added, and after mixing, the mixture was allowed to stand at room temperature for 5 minutes, 3. Mu.L of 1MTris-HCl pH7.4 was added, and after mixing, the supernatant was collected on a magnetic rack.

Supernatant is usedHotSpot Panel v1.0 (Naonda) probe andHybrid Capture Reagents (Naonda) hybrid capture reagent.

As shown in Table 4, the ratio of the amounts of sequencing data of libA and libB was substantially identical to the ratio of the added volumes of the P7U probes. The repetition rate of the two data at the target area is similar, and oversequencing of samples with low initial input and unsaturation of samples with high initial input are avoided.

TABLE 4 actual sequencing of hybridization Pre-library prepared at 1:5

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

From the above description, it can be seen that the above embodiments of the present invention achieve the technical effect that a DNA library with complete sequence can be obtained by using the construction method of the DNA library and using unidirectional PCR amplification and specific probe capture. By adding specific amounts of specific probes, control over the content of DNA fragments in the library can be achieved. In the subsequent mixing of DNA libraries of different sources, the complex steps of respectively quantifying each pre-library, calculating the mixing volume and mixing one by one are not needed, thereby being more beneficial to automation.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method of constructing a DNA library, the method comprising:

a) Connecting a first connector and a second connector at two ends of sample DNA to obtain connector-connected DNA;

b) Using a primer which can be complementarily paired with the first adaptor as a single primer, and carrying out unidirectional PCR amplification on the adaptor-ligated DNA to obtain a unidirectional PCR amplification library;

c) Capturing the unidirectional PCR amplification library by using a capture probe capable of complementarily pairing with the second adaptor to obtain a capture library-probe complex;

d) Removing the capture probes in the capture library-probe complexes to obtain the DNA library.

2. The method of claim 1, wherein using said a) -said c) in said method of construction, different said sample DNA is processed separately to obtain a capture library-probe complex for each of said sample DNA;

mixing the capture library-probe complexes of each of the sample DNA to obtain a mixed sample capture library-probe complex;

removing the capture probes in 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 method according to claim 2, wherein the molar amount of the capture probe added is the same in the method for constructing a sample DNA, and the molar amounts of the different DNA libraries in the mixed sample library are the same.

4. The method of claim 1, wherein the capture probe is a single-stranded DNA, and the capture probe has a modification group for capture.

5. The method of claim 4, wherein the modifying group is a biotin group.

6. The method of claim 4 or 5, wherein magnetic beads capable of capturing the capture probes are added to the capture library-probe complex.

7. The method of claim 6, wherein the magnetic beads comprise streptavidin magnetic beads.

8. The method of claim 6, wherein removing the capture probes in the capture library-probe complexes comprises:

adjusting the pH to be more than or equal to 9 to enable the capture library-probe complex to be subjected to DNA melting, removing the magnetic beads so as to remove the free capture probes, or

And performing PCR amplification on the adaptor-ligated DNA by using a primer capable of complementarily pairing with the second adaptor, and removing the magnetic beads to remove the free capture probes.

9. The method according to claim 8, wherein the capture probe contains uracil, the pH is adjusted to 6-8.5 after removing the magnetic beads, uracil-DNA glycosylase and uracil-specific excision reagent are added to the system, and the remaining fragment of the capture probe is removed.

10. The method of claim 9, wherein the uracil-specific excision reagent is a USER enzyme.

11. A method of constructing a hybrid sample library according to claim 2 or 3, wherein the library of hybrid samples is a pre-hybridization library or a library of sequences to be tested.

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

13. The use according to claim 12, characterized in that it comprises the use in the construction of hybridization pre-libraries or high throughput sequencing libraries.