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Construction method for next-generation sequencing library based on single-stranded connector and application thereof

A sequencing library and construction method technology, applied in the field of biomedicine, can solve problems such as loss of open area information, inability to detect DNA fragment sequences, etc., and achieve the effects of facilitating comparison and analysis, eliminating deviations, and simplifying library construction operations

Active Publication Date: 2018-01-16
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the limitations of the above-mentioned method of library construction based on Tn5 transposome cutting, the use of ATAC-seq technology based on Tn5 transposome cutting chromatin to identify chromatin open regions also has the same disadvantages
Because the library construction method based on Tn5 transposome cleavage cannot detect the sequence of all DNA fragments, ATAC-seq technology will lose part of the open region information due to the library construction process

Method used

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  • Construction method for next-generation sequencing library based on single-stranded connector and application thereof
  • Construction method for next-generation sequencing library based on single-stranded connector and application thereof
  • Construction method for next-generation sequencing library based on single-stranded connector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Based on the SALP of Tn5 transposome incisive reaction

[0061] experimental method:

[0062] Cell culture: HepG2 cells were cultured in DMEM medium. The medium contained 10% (v / v) fetal bovine serum, 100 units / mL penicillin and 100 μg / mL streptomycin. Cells at 37°C, 5% (v / v) CO 2 cultured in an incubator. Cells were obtained from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0063] Genomic DNA preparation: Genomic DNA (gDNA) of HepG2 cells was extracted by phenol-chloroform extraction.

[0064] Adapter preparation: All oligonucleotides were synthesized by Shanghai Sangon (as shown in SEQ ID NO.3-23 in Table 1). Preparation of Tn5 tag adapters (BTAs), tag and ME oligonucleotides with ddH 2 O was dissolved to 20 μM and mixed equimolarly to PCR tubes. Preparation of single-stranded linker (SSA), SSA-PN and SSA-PNre with ddH 2 O was dissolved to 100 μM and mixed equimolarly to PCR tubes. The above o...

Embodiment 2

[0074] Example 2 SALP-seq identification of chromatin opening state in different cell lines

[0075] experimental method:

[0076] Cell culture: HeLa, HepG2 and 293T were cultured in DMEM medium, and GM12878 cells were cultured in RPMI 1640 medium containing 10% (v / v) fetal bovine serum, 100 units / mL penicillin and 100 μg / mL streptomycin. Cells at 37°C, 5% (v / v) CO 2 cultured in an incubator. Cells were obtained from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0077] Adapter preparation: All oligonucleotides were synthesized by Shanghai Sangon (Table 1). Preparation of Tn5 tag adapters (BTAs), tag and ME oligonucleotides with ddH 2 O was dissolved to 20 μM and mixed equimolarly to PCR tubes. Preparation of single-stranded linker (SSA), SSA-PN and SSA-PNre with ddH 2 O was dissolved to 100 μM and mixed equimolarly to PCR tubes. The above oligonucleotide mixture was denatured at 95°C for 5 minutes, and then cooled ...

Embodiment 3

[0091] Example 3 SALP-seq identifies the open state of chromatin through different cell numbers

[0092] experimental method:

[0093] Cell culture: HepG2 was cultured in DMEM medium. The medium contained 10% (v / v) fetal bovine serum, 100 units / mL penicillin and 100 μg / mL streptomycin. Cells at 37°C, 5% (v / v) CO 2 cultured in an incubator.

[0094] Adapter preparation: All oligonucleotides were synthesized by Shanghai Sangon (Table 1). Preparation of Tn5 tag adapters (BTAs), tag and ME oligonucleotides with ddH 2 O was dissolved to 20 μM and mixed equimolarly to PCR tubes. Preparation of single-stranded linker (SSA), SSA-PN and SSA-PNre with ddH 2 O was dissolved to 100 μM and mixed equimolarly to PCR tubes. The above oligonucleotide mixture was denatured at 95°C for 5 minutes, and then cooled down to 25°C naturally.

[0095] Tn5 transposome preparation: According to the instructions of Robust Tn5 Transposase (Robust Tn5 Transposase, Robustnique Corporation Ltd.), mix ...

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Abstract

The invention discloses a construction method for a next-generation sequencing library based on a single-stranded connector and application thereof. The method comprises the following steps: (1) carrying out denaturation on a double-stranded DNA (Deoxyribonucleic Acid) or RNA (Ribonucleic Acid) / DNA heterozygote segment to generate single-stranded DNA; (2) connecting one single-stranded connector with a 3' end of the single-stranded DNA; (3) extending the single-stranded DNA connected with the single-stranded connector by utilizing DNA polymerase to generate double-stranded DNA; (4) connectingthe other end of the double-stranded DNA with a T connector or a Tn5 label connector; (5) carrying out PCR (Polymerase Chain Reaction) amplification on the double-stranded DNA with two ends connectedwith the connectors to form a DNA library capable of sequencing of a next-generation sequencing technology. The method disclosed by the invention can be used for constructing the next-generation sequencing library and determining a DNA sequence and also can be used for identifying a chromatin open region, carrying out gene expression detection, carrying out trace nucleic acid amplification and thelike; the method is a novel method which belongs to the field of nucleic acid detection and analysis and has various functions and wide application value.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for constructing a next-generation sequencing (NGS) library based on Singlestrand Adapter Library Preparation (SALP) and its application. Background technique [0002] Since the emergence of next-generation sequencing (NGS) on the market in 2005, this technology has changed the way we think about scientific research methods in the fields of basic, applied and clinical research. With the continuous development of new methods and computing power, NGS platforms have driven the explosion of biological knowledge in the past few years. As the most important application of NGS, the resequencing of the human genome has greatly deepened our understanding of the relationship between genetic diversity, health and disease. The biggest difference between NGS and traditional Sanger sequencing is that NGS needs to prepare a sequencing library. With the increasing data...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869C40B50/06
Inventor 王进科武剑
Owner SOUTHEAST UNIV
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