阅读说明：本技术 一种低质量dna二代测序文库构建方法 (Method for constructing low-quality DNA next-generation sequencing library ) 是由 杨静 杨俊波 林春艳 曾春霞 于 2020-04-30 设计创作，主要内容包括：本发明公开了一种低质量DNA二代测序文库构建方法,该方法先提取低质量样本中的DNA,然后利用高度降解样本中植物性DNA的检测引物对提取的低质量DNA样本进行多重PCR扩增,确定DNA中是否有植物性DNA；对含有植物性DNA的低质量DNA样本进行建库样品质量检测,检测后根据结果进行样品分类处理,最后采用处理后样品进行低质量DNA的二代测序文库构建,本发明方法操作简便、在保证文库质量的同时减少了试剂的用量,降低了实验成本,大大提高了低质量DNA的二代测序成功率。(The invention discloses a method for constructing a low-quality DNA next-generation sequencing library, which comprises the steps of extracting DNA in a low-quality sample, and performing multiple PCR amplification on the extracted low-quality DNA sample by using a detection primer for highly degrading plant DNA in the sample to determine whether the plant DNA exists in the DNA; the method is simple and convenient to operate, reduces the using amount of reagents while ensuring the quality of the library, reduces the experiment cost, and greatly improves the success rate of second-generation sequencing of low-quality DNA.)

1. A method for constructing a low-quality DNA next-generation sequencing library is characterized by comprising the following steps:

(1) performing multiplex PCR amplification on a low-quality DNA sample extracted by using a detection primer for highly degrading plant DNA in the sample to determine whether the plant DNA exists in the DNA, confirming that the sample contains the plant DNA when one fragment of the amplified 3 items appears, and using the extracted low-quality DNA sample for the next library building experiment;

(2) performing library sample quality detection on a low-quality DNA sample containing plant DNA, performing electrophoresis detection on the low-quality DNA sample by adopting an E-Gel prefabricated agarose Gel electrophoresis system, and simultaneously performing accurate quantification on the low-quality DNA sample by using a Qubit3.0 nucleic acid protein fluorescence quantifier in combination with a Qubit dsDNA HS analysis kit;

(3) detecting a low-quality DNA sample which has no main band but can see a DNA dispersion band and has a sample concentration of more than 0.04 ng/mu L by electrophoresis in the step (2), diluting the sample to 0.04 ng/mu L, taking a diluent to establish a library, detecting the low-quality DNA sample which has no main band, a weak DNA dispersion band or no dispersion band and has low sample concentration and cannot be quantified by a Qubit3.0 nucleic acid protein fluorescence quantifier by electrophoresis in the step (2), and directly establishing the library by using a stock solution;

(4) taking a diluent or stock solution of a 25 mu L low-quality DNA sample to carry out terminal filling and A tail adding reaction, wherein the reaction system is a 30 mu L system;

the reaction system comprises 25 mu L DNA sample, 1.5 mu L Ultra II End Prep Enzyme Mix and 3.5 mu L Ultra II End reaction Buffer, and the reaction conditions are 20 ℃, 30min, 65 ℃, 30min and 4 ℃ for preservation;

(5) performing a linker connection reaction on the reaction product obtained in the step (4), wherein the reaction system is a 46.7 mu L system, and the used linker is NEBNext Adaptor # E6609;

the reaction system comprises the products of the step (3) 30 mu L, Ultra II L alignment Master Mix 15 mu L, L alignment Enhancer 0.5 mu L and 0.6 mu mol/L NEBNext adapter 1.2 mu L, the reaction conditions are 20 ℃ and 15min, the hot cover of a PCR instrument is closed in the process of program execution, and the USER of 1.5 mu L is added into the ligation products immediately after the reaction is finished^TMEnzyme, uniformly mixing, placing at 37 ℃ for 15min, and controlling the temperature of a hot cover of a PCR instrument to be more than or equal to 50 ℃ during program execution;

(6) adding 0.9-1 × AMPure XP Beads magnetic Beads into the reaction product obtained in the step (5) for purification, and using ddH (ddH) to complete purification₂Eluting the purified DNA;

(7) performing PCR amplification enrichment on the purified product in the step (6), wherein the reaction system comprises the purified product in the step (6) 20 mu L, Idex/Universal Primer Mix 5 mu L and Ultra II Q5Master Mix 12.5 mu L;

the reaction conditions comprise pre-denaturation at 98 ℃, 30s, denaturation at 98 ℃, 10s, annealing/extension at 65 ℃, 75s, 16 cycles, final extension at 65 ℃, 5min, storage at 4 ℃, purification of a PCR product by using 0.9-1 × AMPure XP Beads magnetic Beads, and the purified supernatant fluid which is the built library;

(8) the PCR purified product adopts Agilent 2100 BioAnalyzer to detect the length distribution range of the purified product, and the Qubit3.0 nucleic acid protein fluorescence quantitative analyzer is combined with the QubitdsDNA HS analysis kit to accurately quantify the library.

2. The method for constructing a low-quality DNA secondary sequencing library according to claim 1, wherein: the agarose Gel concentration in the E-Gel precast agarose Gel electrophoresis system was 2%.

3. The method for constructing a low-quality DNA secondary sequencing library according to claim 1, wherein: the detection primers for the plant DNA in the highly degraded sample are as follows:

100 bases of primer pairs, the sequence of which is shown as SEQ ID NO: 1 and SEQ ID NO: 2 is shown in the specification;

70 base primer pair, the sequence of which is shown as SEQ ID NO: 3 and SEQ ID NO: 4 is shown in the specification;

a 50-base primer pair, the sequence of which is shown as SEQ ID NO: 5 and SEQ ID NO: and 6.

Technical Field

The invention belongs to the technical field of molecular biology and genomics experiments, and mainly relates to a method for performing second-generation sequencing library building by using DNA in low-quality plant samples (wax leaf specimens and highly degraded plant tissues).

Background

The currently mainstream Sequencing platform mainly comprises Roche/454F L X utilizing pyrosequencing, illuminasequescing systems (HiSeq series, Genome Analyzer IIx, MiSeq) utilizing pyrosequencing, and lifetechnologies Sequencing systems (Applied Biosystems SO L ID and Ion Torque) utilizing ligase Sequencing.

Even if all the second-generation sequencing platforms relate to the library preparation process of a self-made system, the library preparation has certain requirements on the quality of DNA (deoxyribonucleic acid), usually requires a visible obvious main band in agarose gel electrophoresis, has no degradation (or slight degradation), has a DNA concentration of not less than 15 ng/mu L, has high requirements on the initial amount of DNA for library construction, and usually needs 1-0.5 mu g, so that the development and application of the second-generation sequencing technology in more research fields are influenced.

In order to be able to perform scientific research using these low-concentration and low-quality plant DNAs extracted from the above-mentioned materials, it is necessary to improve the existing second-generation sequencing library construction technology and to explore a library construction method suitable for low-quality DNA.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for constructing a low-quality DNA second-generation sequencing library, which provides an effective second-generation library construction method aiming at the highly degraded low-quality plant DNA; the method is simple and convenient to operate, the dosage of reagents is reduced while the library quality is ensured, the experiment cost is reduced, and the success rate of second-generation sequencing of low-quality DNA is greatly improved.

The method for constructing the second-generation sequencing library of the low-quality DNA comprises the following steps:

(1) performing multiple PCR amplification on the extracted low-quality DNA sample by using a detection primer for highly degrading the plant DNA in the sample to determine whether the plant DNA exists in the DNA, so as to judge the pollution degree of the low-quality DNA sample and serve as a basis for performing a next library building experiment; when one amplified 3-item fragment appears, confirming that the sample contains plant DNA, and using the extracted low-quality DNA sample for the next library building experiment;

the detection primer of the plant DNA in the highly degraded sample is derived from the application number 201810111142.3 'detection primer of the plant DNA in the highly degraded sample', and the sequence is as follows:

100 bases of primer pairs, the sequence of which is shown as SEQ ID NO: 1 and SEQ ID NO: 2 is shown in the specification;

70 base primer pair, the sequence of which is shown as SEQ ID NO: 3 and SEQ ID NO: 4 is shown in the specification;

a 50-base primer pair, the sequence of which is shown as SEQ ID NO: 5 and SEQ ID NO: 6 is shown in the specification;

(2) aiming at a low-quality DNA sample, aiming at improving the accuracy of early quality control and better guiding a subsequent library construction experiment, performing library construction sample quality detection on the low-quality DNA sample containing plant DNA, namely performing electrophoresis detection on the low-quality DNA sample by adopting an E-Gel prefabricated agarose Gel electrophoresis system, judging the DNA degradation degree and the fragment size (the DNA of the sample is generally less than 500 bp), and simultaneously performing accurate quantification on the low-quality DNA sample by using a Qubit3.0 nucleic acid protein fluorescence quantifier in combination with a Qubit dsDNA HS (high sensitivity) analysis kit;

(3) detecting a low-quality DNA sample which has no main band but can see a DNA dispersion band and has a sample concentration of more than 0.04 ng/mu L by electrophoresis in the step (2), diluting the sample to 0.04 ng/mu L, taking a diluent to establish a library, detecting the low-quality DNA sample which has no main band, a weak DNA dispersion band or no dispersion band and has low sample concentration and cannot be quantified by a Qubit3.0 nucleic acid protein fluorescence quantifier by electrophoresis in the step (2), and directly establishing the library by using a stock solution;

(4) taking a diluent or stock solution of a 25 mu L low-quality DNA sample to carry out terminal filling and A tail adding reaction, wherein the reaction system is a 30 mu L system;

the reaction system comprises 25 mu L DNA sample, 1.5 mu L Ultra II End Prep Enzyme Mix and 3.5 mu L Ultra II End reaction Buffer, and the reaction conditions are 20 ℃, 30min, 65 ℃, 30min and 4 ℃ for preservation;

(5) performing a linker connection reaction on the reaction product obtained in the step (4), wherein the reaction system is a 46.7 mu L system, and the used linker is NEBNext Adaptor # E6609;

the reaction system comprises the products of the step (3) 30 mu L, Ultra II L alignment Master Mix 15 mu L, L alignment Enhancer 0.5 mu L and 0.6 mu mol/L NEBNext adapter 1.2 mu L, the reaction conditions are 20 ℃ and 15min, the hot cover of a PCR instrument is closed in the process of program execution, and the USER of 1.5 mu L is added into the ligation products immediately after the reaction is finished^TMEnzyme, uniformly mixing, placing at 37 ℃ for 15min, and controlling the temperature of a hot cover of a PCR instrument to be more than or equal to 50 ℃ during program execution;

(6) adding 0.9-1 × AMPure XP Beads magnetic Beads into the reaction product obtained in the step (5) for purification, and using ddH (ddH) to complete purification₂Eluting the purified DNA;

(7) performing PCR amplification enrichment on the purified product in the step (6), wherein the reaction system comprises the purified product in the step (6) 20 mu L, Idex/Universal Primer Mix 5 mu L and Ultra II Q5Master Mix 12.5 mu L;

the reaction conditions comprise pre-denaturation at 98 ℃, 30s, denaturation at 98 ℃, 10s, annealing/extension at 65 ℃, 75s, 16 cycles, final extension at 65 ℃, 5min, storage at 4 ℃, purification of a PCR product by using 0.9-1 × AMPure XP Beads magnetic Beads, and the purified supernatant fluid which is the built library;

(8) the PCR purified product adopts Agilent 2100 BioAnalyzer to detect the length distribution range of the purified product, and the Qubit3.0 nucleic acid protein fluorescence quantitative analyzer is combined with the QubitdsDNA HS analysis kit to accurately quantify the library.

The mass volume concentration of the agarose Gel in the E-Gel precast agarose Gel electrophoresis system is 2%.

The invention has the advantages and technical effects that:

the method can utilize the low-quality plant DNA which is highly degraded and has extremely low concentration (the concentration of the DNA sample is less than or equal to 0.04 ng/mu L) and can not be used by the conventional method to complete the construction of a second generation sequencing library under the condition of using as few reagents as possible in a short working time, and has high success rate.

The method is simple and convenient to operate, the dosage of the reagent is reduced while the library quality is ensured, the experiment cost is reduced, and the success rate of second-generation sequencing of low-quality DNA is greatly improved.

Drawings

FIG. 1 shows the detection results of the amplification products of multiplex PCR.

Detailed Description

The present invention will be described in further detail with reference to examples; the following examples are given solely for the purpose of illustration and are not to be construed as limitations of the present invention, as many insubstantial modifications and variations of the present invention are possible to those skilled in the art in light of the above teachings; reagents used in the construction method of the next generation sequencing library provided by the invention can be purchased from the market; 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.