CN111363784A - Method and kit for constructing sequencing library - Google Patents

Abstract

The application provides a method for constructing a DNA library and a kit for constructing the DNA library. The method and the kit generate DNA fragments with the length of 450bp or less by mixing viral RNA, reverse transcriptase and a specific primer pair to perform reverse transcription PCR (RT-PCR) and PCR; and using ligase to connect the DNA fragment, barcode (barcode) and linker to generate a DNA library simply and rapidly. Compared with the traditional library building process, the method reduces the steps of segment cutting and interruption, saves time and increases efficiency. Meanwhile, compared with a method for fusing primers, the method avoids the problems of non-specific amplification, low-efficiency amplification and the like caused by overlong length of the fused primers, and has the advantages of more flexibility and reliability. The kit simplifies the experimental steps by directly amplifying the region to be sequenced through the RT-PCR and the PCR of the specific region primer one-step method, thereby effectively improving the construction of the DNA library applied to the second-generation sequencing platform.

Description

Method and kit for constructing sequencing library

Technical Field

The application relates to the technical field of sequencing, in particular to a method and a kit for constructing a DNA library of a second-generation sequencing platform.

Background

In 1977, the dideoxy chain termination method DNA sequencing method, also known as Sanger method, was invented by Fredrichek Sanger, UK. The dideoxy chain termination method and the chemical degradation method and the derivative method thereof are collectively called the first generation DNA sequencing technology, and are the main sequencing method used by the human genome project. But it has been gradually eliminated due to its disadvantages of high cost, long detection period, low flux, etc.

High-throughput Sequencing is a revolutionary change to traditional Sanger Sequencing and is referred to as Next Generation or Next Generation Sequencing (NGS) because it allows Sequencing of hundreds of thousands to millions of nucleic acid molecules at a time. The high-throughput sequencing technology has the characteristics of realizing multi-gene parallel detection, having higher accuracy and sensitivity than the traditional detection method, and being capable of more truly detecting the virus or tumor mutation panorama. With the rapid development of second generation sequencing technologies typified by HiSeq X-10, MiSeq and Nextseq 500, more and more biological problems are solved by second generation sequencing technologies. The application in the aspect of medication guidance is wide, wherein the most important is guidance of virus resistance, gene detection of tumor individualized treatment and the like. Compared with the traditional detection, the NGS technology can more fully cover the gene variation condition and provide more references for individualized medication of patients.

In the NGS platform, Ion Torrent is the first NGS platform to use no optical sensing. The principle of Ion Torrent is to break the extracted DNA into fragments with certain length before sequencing, and then connect the single fragments with a micro-bead full of probes. Then, the probes distributed on the surface of the microsphere are amplified into DNA fragments with the same sequence by an amplification technology, and the microsphere is like a hair ball. Thereafter, the microspheres were placed on a sequencing chip containing millions of microwells and at least one DNA synthesis occurred by placing a liquid containing one base at a time, repeating each cycle 4 times to place a different base (A \ T \ G \ C). During synthesis, dntps release hydrogen ions, which change the pH, and the signal present at a certain base in a certain round is detected by a sensor such as a complementary metal-oxide-semiconductor (CMOS) and ion-sensitive field-effect transistor (ISFET) at the bottom of the microwell and converted into an electrical signal. Finally, the actual sequence of the DNA can be obtained by integrating the electrical signal.

The traditional method for constructing the amplicon library in the NGS platform has complicated steps, and needs to perform PCR amplification, enzymolysis and digestion, joint connection, repeated purification and other steps for many times. Not only does the conventional NGS platform cost a significant amount of time and personnel, but the library is susceptible to contamination due to the need to repeatedly expose the sample to air in a multi-step operation. In addition, increasing library manipulation steps results in high loss rates and reduced library complexity, which reduces the detection rate of low abundance mutations. In addition, in the traditional method for constructing the amplicon library, the cost for establishing the library by a single sample is high, and the clinical application of the NGS technology is seriously influenced.

In addition, construction of NGS libraries by primer fusion is widely practiced. However, this method requires the synthesis of a sequencing linker and a barcode in advance, which not only increases the length of the primer, but also may cause disadvantages such as non-specific amplification and reduction in amplification efficiency.

The present application aims to provide a kit for constructing a DNA library in a one-step method, and a method for constructing a DNA library by operating the kit. Through the kit and the method for constructing the DNA library, the advantages of a PCR amplification sequencing region can be utilized, different linkers and barcode are adopted to be directly connected with a PCR product, the sequencing flexibility is increased, and the virus DNA library for an ion torrent second-generation sequencing platform can be efficiently prepared.

Disclosure of Invention

The purpose of the present application is to provide a method for constructing a DNA library, and a kit for constructing a DNA library.

The above object of the present invention is achieved by the following technical means.

A method of constructing a DNA library, comprising:

mixing viral RNA, reverse transcriptase and a specific primer pair to perform reverse transcription PCR (RT-PCR) and PCR to generate a DNA fragment; and

ligating the DNA fragments, barcodes (barcodes) and linkers using a ligase to generate a DNA library;

the primer pair comprises at least one primer pair selected from the group consisting of an upstream primer and a downstream primer as shown in the following table;

preferably, the length of the DNA fragment is below 450 bp.

Preferably, the ligase comprises T4 ligase.

Preferably, the RT-PCR reaction is carried out at 45-55 ℃ for 15-45 minutes, at 90-100 ℃ for 1.5-2.5 minutes; the PCR reaction is carried out for 25-35 cycles of 15-45 seconds at 90-100 ℃, 15-45 seconds at 50-60 ℃ and 1.5-2.5 minutes at 65-80 ℃.

Preferably, the ligation step is performed at 20-37 ℃ for 5-40 minutes.

Preferably, the virus comprises HIV.

Preferably, the method further comprises: after the reverse transcription PCR and PCR, the DNA fragment was purified using magnetic beads.

A kit for constructing a DNA library, comprising a reverse transcriptase and a specific primer pair;

the primer pair comprises at least one primer pair selected from the group consisting of an upstream primer and a downstream primer as shown in the following table;

preferably, the kit further comprises a ligase, a barcode (barcode), and a linker.

Preferably, the kit further comprises magnetic beads.

Drawings

FIG. 1 illustrates the principle and operational flow of a construction method according to an embodiment of the present application.

FIG. 2 shows the DNA fragments amplified after RT-PCR and PCR by specific primers.

FIGS. 3 and 4 show the distribution of DNA fragment lengths in the library as measured by an Agilent 2100 Bioanalyzer.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In the event that a definition used herein conflicts or disagrees with a definition contained in another publication, the definition used herein shall govern.

As used herein, the terms "selected from", "consisting of …" (compounded of) are synonymous with "comprising". As used herein, the terms "comprises," "comprising," "includes," "including," "has," "contains," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a plurality of elements listed in a list is not necessarily limited to only those elements listed in the list, but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

When an amount, concentration, or other value or parameter is given as either a range, preferred range, or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range is recited as "1 to 5," the recited range should be understood to include the ranges "1 to 4," "1 to 3," "1 to 2 and 4 to 5," "1 to 3 and 5," and so forth. If a range of values is recited in this specification, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range.

Furthermore, unless expressly stated otherwise, "or" means an inclusive "or" and not an exclusive "or". For example, the condition a "or" B "satisfies any one of the following cases: a is true (or present) and B is false (or not present), a is false (or not present) and B is true (or present), and both a and B are true (or present).

Similarly, the indefinite articles "a", "an" and "an" preceding an element or ingredient herein are intended to illustrate, without limitation, the number of instances (i.e., occurrences) of the element or ingredient, unless the context clearly indicates otherwise. Thus, "a", "an" and "the" are to be understood as including one or at least one, and the singular forms of the elements or components also include the plural.

The present application is an improved method and kit based on the method of constructing DNA libraries in Ion Torrent sequencing platform, performing formulation mixing and simplification steps. The operation principle and flow of the method for constructing DNA library of the present application are shown in FIG. 1. In one aspect of the present application, a method of constructing a DNA library comprises: mixing viral RNA, reverse transcriptase and a specific primer pair to perform reverse transcription PCR (RT-PCR) and PCR to generate a DNA fragment; and ligating the DNA fragment, a barcode (barcode) and a linker using a ligase to generate a DNA library. To achieve the purpose of simplifying the procedure, the present application mixes viral RNA, reverse transcriptase and specific primer pair in one step to perform reverse transcription PCR (RT-PCR) and PCR to directly generate DNA fragment. Compared with the prior method, the method of the one-step method is simpler and more convenient to operate, and can reduce the loss of samples and increase the integrity of the library.

The amplicon library used to construct the target gene of the viral reverse transcription cDNA sample is based on the characteristics of the NGS sequencing method. Because sequencing of NGS has strict requirements on the length of a fragment, the method strictly controls the length of a DNA fragment (namely an amplicon) to be below 450bp by means of primer design. In one embodiment of the present application, the DNA fragment has a length of 450bp or less, preferably 350bp-420 bp.

In order to make the length of the DNA fragment (i.e., amplicon) less than 450bp, the present application obtains specific primers by design. In one embodiment of the present application, said primer pair for Pol gene of target gene comprises at least one primer pair selected from the group consisting of an upstream primer and a downstream primer as shown in the following table;

because sequencing of NGS has strict requirements on the length of the fragment, if the DNA fragment of the target gene is too long, primers can be designed in an overlapping mode, so that the aim of covering all gene regions needing sequencing in a large range is fulfilled.

The primer sequences for PCR include only the amplification region, and do not include the sequencing linker sequence and the barcode sequence. Can increase the PCR amplification efficiency to the maximum extent and reduce the non-specific amplification. After PCR is completed, one or more PCR products (different in primer sequence) from the same sample are mixed, and ligation reaction library construction is completed by ligase. After the library is built, different sample mixed sequencing can be carried out according to the barcode. In one embodiment of the present application, the ligase comprises T4 ligase. In one possible embodiment, the ligase may be any other ligase without affecting the effectiveness of the ligase.

In the method for constructing the DNA library, in order to ensure that the sample RNA and the reverse transcriptase can properly react to successfully carry out reverse transcription to form DNA and the DNA directly reacts with the specific primer pair to successfully generate a DNA fragment, the RT-PCR reaction is carried out for 15-45 minutes at 45-55 ℃ and 1.5-2.5 minutes at 90-100 ℃; the PCR reaction is carried out for 25-35 cycles of 15-45 seconds at 90-100 ℃, 15-45 seconds at 50-60 ℃ and 1.5-2.5 minutes at 65-80 ℃. Preferably at 50 ℃ for 30 minutes and at 94 ℃ for 2 minutes; 30 cycles of 30 seconds at 94 ℃, 30 seconds at 55 ℃ and 2 minutes at 72 ℃.

In the method for constructing a DNA library of the present application, in order to ligate the DNA fragments and the adaptors by using ligase to generate a DNA library, the temperature of the ligation step is generally 20 to 30 ℃, preferably 25 ℃ depending on the enzyme used. The time for the ligation step is between 5 and 20 minutes, preferably 15 minutes. After the reaction is complete, the reaction is terminated at a temperature of 65 to 75 deg.C, preferably 72 deg.C, for a period of 3 to 10 minutes, preferably 5 minutes.

The method and kit for constructing a DNA library of the present application can be applied to viruses. If the genetic information of the virus is stored in deoxyribonucleic acid (DNA), the DNA of the virus can be directly extracted and used as sample DNA to perform the method described in the present application. If the genetic information of the virus is stored on ribonucleic acid (RNA), the methods described herein can be performed by extracting the RNA of the virus using reverse transcriptase and random primers to generate cDNA as sample DNA. In one aspect of the application, the method of constructing a DNA library further comprises reverse transcribing the viral RNA into cDNA. The cDNA is used as sample DNA, and a cutting step of mixing with an enzyme composition is carried out.

Viruses that can be used in the present application as indicated above include, but are not limited to, HIV (human immunodeficiency virus), Hepatitis C virus (Hepatitis C virus), Japanese Encephalitis Virus (JEV), Influenza virus (Influenza A virus), rhinovirus (rhinovirus), poliovirus (poliovirus), coxsackievirus (coxsackievirus), Dengue virus (Denv), Rotavirus (Rotavirus, RV), Tobacco mosaic virus (Tobacco mosaic virus, TMV), severe acute respiratory syndrome coronavirus (lung acute respiratory syndrome coronavirus, SARS-CoV), Middle East respiratory syndrome coronavirus (Middorsyndrome coronavirus, Marburg), Marburg virus (Marburg virus), and Marburg virus (Marburg virus). In one embodiment of the present application, the virus comprises HIV-1 or HIV-2.

The DNA fragments obtained by RT-PCR and PCR in the present application can be purified as required before the ligation step. In one embodiment of the present application, the DNA fragments are purified using magnetic beads.

The method of DNA library construction as shown above is carried out using, but not limited to, the kit for DNA library construction of the present application. The kit for constructing a DNA library of the present application comprises a reverse transcriptase and a specific primer pair to reverse transcribe RNA of a virus into cDNA for re-amplification for use in the kit of the present application. In one embodiment of the present application, the kit further comprises a ligase, a barcode (barcode), and a linker, through which the barcode (barcode), the linker and the DNA fragment are ligated. In one embodiment of the present application, the kit further comprises magnetic beads, and the DNA fragments are purified with the magnetic beads before the ligation step.

The quality of the DNA library obtained by the method for constructing the DNA library or the kit for operating the DNA library can be further quantitatively and qualitatively detected. In one embodiment of the present application, the DNA library can optionally be quantified, but not limited to, by Real-time PCR or by using fluorescent dyes. In one embodiment of the present application, the range of fragment distribution in a DNA library can be detected, optionally but not limited to, by agarose gel electrophoresis or capillary electrophoresis systems.

Examples

The invention claimed in this application is further illustrated in the following detailed description. These examples are intended only to illustrate the invention claimed herein and are not intended to limit the scope of the invention claimed herein. Further, various changes or modifications may be made by one skilled in the art to the invention, and equivalents may fall within the scope of the invention as defined by the claims appended hereto.

Conversion of RNA to DNA and amplification by RT-PCR and PCR

Verso1-Step RT-PCRKittReddyMix (ThermoFisher (China)) was used, and the components contained in the RT-PCR and PCR mixture, such as reverse transcriptase, specific primers, and the ratio thereof are shown in Table 1 below.

TABLE 1

System of	Volume (μ L)
		Verso Enzyme mixture (Verso Enzyme Mix)	0.4
2X1 Step polymerase chain reaction premix (2X1-Step PCR Ready Mix)	10
		Reverse transcription Enhancer (RT Enhancer)	1
Positive primer (Forward primer) 20. mu.M	1
		Reverse primer (Reverse primer) 20. mu.M	1
Nuclease-free Water (Nuclear-free Water)	1.6
		Totaling:	15

RT-PCR and PCR mix were mixed with 5. mu.L of RNA extracted from HIV-1, making the whole reaction system 20. mu.L, by reaction conditions: 50 ℃ for 30 minutes; 94 ℃ for 2 minutes; 30 cycles at 94 ℃, 30 seconds, 55 ℃, 30 seconds, and 72 ℃ for 2 minutes; 72 ℃ for 10 minutes; DNA fragments were generated at 4 ℃ for 1 minute.

From the electrophoresis results of FIG. 2, it can be seen that the lengths of the DNA fragments obtained by RT-PCR and PCR through the specific primer pair of the present application are nearly identical, and the DNA fragments are located at about 300bp, which is in accordance with the interval required by the present application.

DNA fragment purification

A mixture of magnetic beads as shown in Table 2 below was prepared by mixing a magnetic bead purification reagent AgencourtAmpure XP (Backman Coulter trade (China) Co., Ltd.) with DNA fragments.

TABLE 2

The mixture of magnetic beads was purified by passing through 1.8 × beads, washing 2 times with 200. mu.L of 70% ethanol, and eluting with 25. mu.L of Low TE to obtain a purified DNA fragment.

DNA fragment connection joint

The linker was Ion XPress Barcode Adaptors 1-16Kit (ThermoFisher (China) Co., Ltd.), the Ligase was T4 DNA Ligase (New England Biolabs (Beijing) Co., Ltd.), and Bst 2.0

DNA Polymerase, purified DNA fragments were mixed to prepare ligation mixtures as shown in Table 3 below.

TABLE 3

Subjecting the ligation mixture to reaction conditions of 25 ℃ for 15 minutes; and 5 min at 72 ℃, obtaining a DNA fragment connected with the joint, and completing the construction of the DNA library. The DNA library was stored at 4 ℃ for subsequent sequencing steps.

DNA library quantification and quality detection

A quantitative Kit Ion Library TaqMan quantification Kit (ThermoFisher (China) Co., Ltd.) was used. The E.coli DH10B Control Library was diluted in gradient at 0.1, 0.01, 0.001, 0.0001, 0.00001, respectively, and at 6.8pM, 0.68pM, 0.068pM, 0.0068pM, 0.00068pM, respectively. The sample to be quantified is diluted in a gradient way, and the dilution ratio is 1: 20, 1: 2000 and 1: 20000 respectively. qPCR mixtures were prepared and the like are as in table 4 below.

TABLE 4

Mixing the qPCR mixture with 5 μ L of sample DNA or standard substance diluted in gradient to make the whole reaction system be 20 μ L, and allowing the reaction conditions to be 50 deg.C and 2 min; 95 ℃ for 20 seconds; the resulting DNA library was quantified at 95 ℃ for 1 second and at 60 ℃ for 20 seconds for 40 cycles. The concentration was calculated from the Ct value of real-time fluorescent quantitative PCR. The sample concentration was calculated as follows:

sample concentration-concentration × dilution by qPCR assay

The final concentration of each sample was quantified to a certain value between 25-60pM, and after mixing, 25. mu.L of each sample was used to detect the fragment length distribution in the DNA library with a 2100Bioanalyzer microfluidic electrophoresis analyzer (Agilent 2100Bioanalyzer), the results are shown in FIGS. 3 and 4. From the electrophoresis results of FIG. 3, a band pattern was observed around 300bp, indicating that a DNA fragment of 300bp in length was present. From the fragment length distribution of FIG. 4, signals appear at 35bp, 150 bp, 500bp, 10380 bp. Wherein the signal amplitude of 150-500bp is large, which means that the length of the obtained fragment is more than 150-500 bp. From the results of quantitative and quality detection of the DNA library, the method and the kit of the present application can construct a DNA library with good quality, which is suitable for the subsequent steps of the second generation sequencing.

Although the invention has been described with reference to the above embodiments, it should be understood that the invention is not limited thereto, and those skilled in the art can make modifications and combinations of the above embodiments without departing from the spirit and scope of the invention.

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Claims (10)

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

mixing viral RNA, reverse transcriptase and specific primer pair to perform reverse transcription PCR (RT-

PCR) and PCR, to generate DNA fragments; and

ligating the DNA fragments, barcodes (barcodes) and linkers using a ligase to generate a DNA library;

the primer pair comprises at least one primer pair selected from the group consisting of an upstream primer and a downstream primer as shown in the following table;

numbering Upstream primer Downstream primer 1 SEQ ID No：1 SEQ ID No：2 2 SEQ ID No：3 SEQ ID No：4 3 SEQ ID No：5 SEQ ID No：6 4 SEQ ID No：7 SEQ ID No：8 5 SEQ ID No：9 SEQ ID No：10 6 SEQ ID No：11 SEQ ID No：12 7 SEQ ID No：13 SEQ ID No：14 8 SEQ ID No：15 SEQ ID No：16 9 SEQ ID No：17 SEQ ID No：18 10 SEQ ID No：19 SEQ ID No：20 11 SEQ ID No：21 SEQ ID No：22

。

2. The method of claim 1, wherein the DNA fragment is less than 450bp in length.

3. The method of claim 1, wherein the ligase comprises T4 ligase.

4. The method of claim 1, wherein the RT-PCR reaction is performed at 45-55 ℃ for 15-45 minutes, at 90-100 ℃ for 1.5-2.5 minutes; the PCR reaction is carried out for 25-35 cycles of 15-45 seconds at 90-100 ℃, 15-45 seconds at 50-60 ℃ and 1.5-2.5 minutes at 65-80 ℃.

5. The method of claim 1, wherein the ligating step is performed at 20-37 ℃ for 5-40 minutes.

6. The method of claim 1, wherein the virus comprises HIV.

7. The method of claim 1, further comprising: after the reverse transcription PCR and PCR, the DNA fragment was purified using magnetic beads.

8. A kit for constructing a DNA library, comprising a reverse transcriptase and a specific primer pair;

the primer pair comprises at least one primer pair selected from the group consisting of an upstream primer and a downstream primer as shown in the following table;

numbering Upstream primer Downstream primer 1 SEQ ID No：1 SEQ ID No：2 2 SEQ ID No：3 SEQ ID No：4 3 SEQ ID No：5 SEQ ID No：6 4 SEQ ID No：7 SEQ ID No：8 5 SEQ ID No：9 SEQ ID No：10 6 SEQ ID No：11 SEQ ID No：12 7 SEQ ID No：13 SEQ ID No：14 8 SEQ ID No：15 SEQ ID No：16 9 SEQ ID No：17 SEQ ID No：18 10 SEQ ID No：19 SEQ ID No：20 11 SEQ ID No：21 SEQ ID No：22

。

9. The kit of claim 8, further comprising a ligase, a barcode (barcode), and a linker.

10. The kit of claim 8, further comprising magnetic beads.

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