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A DNA sequencing method and a next-generation sequencing library

A sequencing library and sequencing technology, applied in the field of molecular biology, can solve the problems of directly distinguishing the requirement of the amount of starting DNA template for target single-stranded DNA, and the requirement of lower amount of starting DNA template

Active Publication Date: 2019-01-22
SHANGHAI MAJORBIO BIO PHARM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The present invention provides a DNA sequencing method for the defects in the prior art that the sense strand and the antisense strand of the target single-stranded DNA cannot be directly distinguished through the sequencing results and the requirement for a high amount of starting DNA template , this method can not only directly distinguish the original template sequence of the target single-stranded DNA through the sequencing results, but also has low requirements for the amount of the starting DNA template

Method used

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  • A DNA sequencing method and a next-generation sequencing library
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  • A DNA sequencing method and a next-generation sequencing library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1D

[0049] Example 1DNA sample preparation

[0050] The sample to be prepared is viral single-stranded DNA, and the QIAamp UltraSens Virus Kit (QIAGEN) is used for DNA extraction. Proceed as follows:

[0051] 1) Cultivate single-stranded DNA virus (Canine parvovirus canine parvovirus) with NL-DK1 cells in 5% fetal bovine serum, take 1ml of the culture solution into a 2ml EP tube, then add 0.8ml Buffer AC, add 5.6μl carrier RNA, mix;

[0052] 2) The mixed sample was left at room temperature for 10 minutes;

[0053] 3) Centrifuge at 1200×g for 3 minutes, discard the supernatant;

[0054] 4) Add 300 μl 60°C preheated Buffer AR and 20 μl proteinase K, and mix until the precipitate is completely suspended in the liquid;

[0055] 5) Incubate at 40°C for 10 minutes, and mix the samples every 5 minutes;

[0056] 6) Add 300μl Buffer AB and mix well;

[0057] 7) Add the mixed liquid to the adsorption column, centrifuge at 3000-5000×g for 1 minute, discard the waste liquid, and transfe...

Embodiment 2 2

[0062] Example 2 Construction and sequencing of next-generation sequencing library

[0063] Rationale for Sequencing Methods figure 1 .

[0064] 1) Complementary extension:

[0065] Add the following reaction system to the reaction tube:

[0066]

[0067]

[0068] Carry out the following reaction procedure: 1-5min at 95°C; 10min at 25°C; pause at 37°C, and continue the reaction after adding the following reagents;

[0069] Add 0.5 μl of Klenow (NEB) to the reaction tube;

[0070] The following reaction procedure was carried out: 37°C for 30 minutes; 70°C for 10 minutes; and 10°C storage to obtain a reaction product containing a complementary strand of viral single-stranded DNA linked with a specific sequence.

[0071] The above-mentioned DNA added to the reaction tube is the viral single-stranded DNA prepared in Example 1, and the above-mentioned Read2-NB-1 (SEQID NO: 4) is a random primer complementary to the viral single-stranded DNA, and the nucleotide sequence is...

Embodiment 3

[0109] Example 3 Analysis of sequencing results

[0110] 1) The single-stranded DNA sequencing library constructed in Example 2 was sequenced on the Illumina sequencing platform, 251 cycles;

[0111] 2) Using the above method, the following results were obtained: a total of 7073 pairs of sequences were obtained, and after removing repeated sequences and low-quality sequences, a total of 6893 pairs of high-quality sequences (that is, obtained after removing repeated sequences and sequences whose quality did not reach Q20 sequence with unique features);

[0112] 3) The obtained high-quality sequence was assembled using the software SOAPdenovo v2.04 (http: / / soap.genomics.org.cn / ), and a contig was obtained, the contig N50 was 5323 (genome size was 5323bp), and the obtained sequence was sequenced Covers the entire genome.

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Abstract

The invention discloses a DNA sequencing method and a next generation sequencing library. The sequencing method comprises the steps of mixing a DNA fragment to be measured with a random primer Read2-NB, conducting an extension reaction, connecting the 3' end of an obtained extension product with a chain b which is in a double chain joint Read1-Adapter and contains a sequencing platform specific sequence, and taking amplification connection products of specific primers T1 and T2, so that construction of the next generation sequencing library is completed, and subsequently, conducting sequencing. By means of the DNA sequencing method and the next generation sequencing library, library construction and sequencing can be conducted on double chain DNA and / or single chain DNA, the amount of required initial DNA is lowered, applicability of limited samples for template DNA is great, and the original template sequence of the target single chain DNA can be directly distinguished out of the sequencing result.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to a DNA sequencing method and a next-generation sequencing library. Background technique [0002] The genetic material of life mainly exists in the DNA double helix structure, and a small amount exists in the structure of single-stranded DNA, single-stranded RNA, and double-stranded RNA. In order to uncover the genetic information of life, various forms of genetic material need to be sequenced, that is, gene sequencing of single-stranded DNA, double-stranded DNA, single-stranded RNA, and double-stranded RNA. [0003] Double-stranded DNA will temporarily remain in the form of single-stranded DNA during replication, damage repair, denaturation, or other specific treatments, and relevant researchers need to perform sequencing studies on this specific sequence. In addition, organisms whose genetic material is single-stranded DNA are mainly viruses. As of September 6, 2015, 827 single...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6869C40B40/06C40B50/06
CPCC12Q1/6869C40B40/06C40B50/06
Inventor 于丹张祥林林芹马旖蓵史彩萍
Owner SHANGHAI MAJORBIO BIO PHARM TECH
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