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Building method of long fragment nucleic acid library

A construction method and nucleic acid library technology, which is applied in the field of long-fragment nucleic acid library construction, can solve problems such as difficulty in assembly, and achieve the effects of improving assembly accuracy and shortening assembly time

Inactive Publication Date: 2015-06-17
BIOMARKER TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a long-fragment nucleic acid library construction method to solve the current problems of difficult assembly caused by factors such as polyploidy, high heterozygosity, and repetitive sequences for complex genome assembly

Method used

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  • Building method of long fragment nucleic acid library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1 Long fragment nucleic acid library construction method

[0071] (1) Preparation of large fragment library

[0072] ① Genomic DNA Fragmentation

[0073] 20μg (Nanodrop quantitative) genomic DNA samples of rice Nipponbare were crushed by nebulization to obtain diffuse bands with concentrated bands of 5K and 6K, and the 5K and 6K fragments were recovered respectively. For electrophoresis detection, see figure 1 ;

[0074] ②End repair, adding A, adding joints

[0075] The purified large fragments were end-repaired with T4 DNA polymerase, T4PNK and Klenow enzyme and purified with a PCR product purification kit. After end repair and purification, the purified sample was treated and purified with A, and T4 DNA ligase was used to add a linker. The linker was MACCA (the NNN fixed position CCA in MANNN, to verify the linker connection efficiency), synthesized by Invitrogen, and the sequence was:

[0076] F: '-ACTTCCATCCCCATCCCCCATCCCCCATCCCGCTCTTCCGATCT;

[0077...

Embodiment 2

[0121] Example 2 Long Fragment Nucleic Acid Library Construction Method and Sequence Assembly

[0122] (1) Preparation of large fragment library

[0123] ① Genomic DNA Fragmentation

[0124] Take 20μg (Nanodrop quantification) of rice Nipponbare genomic DNA sample, sample number is C27, use the atomization method to break up the genomic DNA, obtain a diffuse band with a concentrated band of 10K, and recover the 10K fragments respectively. For the electrophoresis detection, see Figure 6 ;

[0125] ②End repair, adding A, adding joints

[0126] The purified large fragments were end-repaired with T4 DNA polymerase, T4PNK and Klenow enzyme and purified with a PCR product purification kit. After end repair and purification, the purified sample is treated and purified with A, and T4 DNA ligase is used to add a linker. The linker is MANNN, synthesized by Invitrogen, and the sequence is:

[0127] F:'-ACTTNNNTCCCNNNTCCNNNTCCNNNTCCCCGCTCTTCCGATC T;

[0128] R: 5'-GATCGGAAGAGCACACGT...

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Abstract

The invention provides a building method of a long fragment nucleic acid library. The method comprises the following steps: preparing a batch of Index label clusters, marking long fragment nucleic acid molecules in batch, using whole genome resequencing library building to build long fragment libraries suitable for illumine sequencing platforms, allowing all fragments to have connectors suitable for the illumine sequencing platforms under the action of transposase, carrying out PCR amplification, and screening the well build library. The method effectively solves the problem of influences of polyploid and repeated sequence in a genome de novo sequencing technology on the assembling accuracy, and can be used for assembling complex genomes and assisting de novo sequencing assembling to improve the assembling accuracy and shorten the assembling time.

Description

technical field [0001] The invention relates to the field of bioinformatics, in particular to a method for constructing a long fragment nucleic acid library suitable for genome de novo sequencing and assembly technology. Background technique [0002] High-throughput sequencing technology, also known as next-generation sequencing technology, is a revolutionary change compared to traditional sequencing technology, which can simultaneously sequence millions of DNA molecules. High-throughput sequencing technology can not only perform large-scale genome sequencing, but also be used for gene expression analysis, identification of non-coding small molecule RNA, screening of transcription factor target genes and DNA methylation and other related research. [0003] Whole-genome de novo sequencing is also called genome de novo sequencing, which refers to sequencing the genome of a species without relying on any known genome sequence information, and then applying bioinformatics method...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68C40B50/06
Inventor 郑洪坤刘少卿
Owner BIOMARKER TECH
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