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Method for establishing DNA (Deoxyribose Nucleic Acid) library based on illumina sequencing platform

A DNA library and construction method technology, which can be applied to libraries, chemical libraries, nucleotide libraries, etc., can solve the problems of limited number of clones, long cycle and high cost, and achieve the effect of shortening construction time and expanding construction scope.

Inactive Publication Date: 2014-11-19
SHANGHAI MAJORBIO BIO PHARM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But NxSeq TM The 40 kb Mate-Pair Cloning Kit needs to construct a fosmid library, which is not only costly and takes a long time, but also the number of clones obtained is limited, which is not conducive to the construction and sequencing of large-scale libraries

Method used

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  • Method for establishing DNA (Deoxyribose Nucleic Acid) library based on illumina sequencing platform
  • Method for establishing DNA (Deoxyribose Nucleic Acid) library based on illumina sequencing platform
  • Method for establishing DNA (Deoxyribose Nucleic Acid) library based on illumina sequencing platform

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Construction of 20K Large Fragment Library of Turbot Genomic DNA

[0057] 1. Sample DNA interruption:

[0058] First take about 1 ug of sample DNA and break it with a HydroShear large knife head (the speed parameter is set to 15), and use 0.5% agarose gel to detect whether the fragment size is around 20K. If the segment size is not appropriate, adjust the speed code and try again until a suitable interruption condition is selected. The turbot genomic DNA uses this interruption condition, and the DNA fragments are concentrated around 20K, which meets the experimental requirements. Continue to use this condition to interrupt the 20ug DNA sample.

[0059] 2. DNA end filling:

[0060] 1) Prepare the following mixture in a PCR tube:

[0061]

[0062] 2) Gently mix and place at 20°C for 30 min.

[0063] 3) Transfer the DNA into a 1.5ml centrifuge tube, add 200μl Ampure XP beads, mix gently, and place at room temperature for 15min.

[0064] 4) The sample was ...

Embodiment 2

[0170] Example 2 Evaluation of the MP library of the turbot genomic DNA 20K large fragment library

[0171] MP library evaluation was performed on the 20K large fragment library of turbot genomic DNA constructed in Example 1 above.

[0172] The evaluation of the MP library mainly has two important indicators: the average library length that matches the library construction expectation and the ratio of the read pairs with the insert size in the normal range (ie, the circularization rate), which are two important factors to determine the success of the experiment. standard.

[0173] MP library evaluation process: Deduplicate and quality control the sequencing read pairs, then use the software Bowtie2 to compare to the assembled scaffold sequence, use the script written by yourself to compare and compare the results for data statistics, and obtain the statistical values ​​in Table 1:

[0174] Table 1 Evaluation results of 20K-MP library

[0175]

[0176]

[0177] Note: Th...

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Abstract

The invention relates to the technical field of high throughput sequencing, and discloses a method for establishing a DNA (Deoxyribose Nucleic Acid) library based on an illumina sequencing platform. The method comprises the following steps: (1) randomly interrupting the genome DNA; (2) filling the tail ends, and adding biotin labeled cyclizing connectors at two ends; (3) separating DNA fragments, wherein the sequence of the step (2) and the step (3) can be changed;; (4) cyclizing, and removing uncyclized DNA fragments; (5) randomly interrupting cyclized DNA; (6) separating biotin labeled DNA fragments; (7) filling the tail ends of the biotin labeled DNA fragments; (8) adding an A alkali group; (9) connecting with the sequencing connectors; (10) performing PCR amplification and enrichment, thereby obtaining the sequencing library. The method adopts a Cre recombinase system to cyclize the DNA fragments, the cyclizing only takes 1 hour, the time for establishing the library is greatly shortened, the large-fragment DNA library is successfully established, and splicing of genomes in sequencing can meet the standard of a fine diagram or accomplished diagram.

Description

technical field [0001] The invention relates to the technical field of high-throughput sequencing, in particular to a method for constructing a large fragment DNA library based on a high-throughput sequencing platform. Background technique [0002] High-Throughput Sequencing (High-Throughput Sequencing), also known as Next Generation Sequencing (NGS), is relative to traditional Sanger Sequencing (Sanger Sequencing). At present, there are three major platforms for high-throughput sequencing: Roche's 454 sequencer, Illumina's Hiseq / Miseq sequencer and ABI's SOLiD sequencer. The most widely used is the illumina sequencing platform. Although high-throughput sequencing has a large amount of data and a low cost per unit of data, its read length is shorter than that of Sanger sequencing. The longest read lengths of Miseq and Hiseq currently provided by Illumina are 2x300bp and 2x150bp, respectively. The short read length makes the assembly of de novo sequencing (De Novo Sequencin...

Claims

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Application Information

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IPC IPC(8): C40B50/06C40B40/06C12Q1/68
Inventor 王真艳陈昌岳林爱萍李静张祥林胡秋萍陶晔
Owner SHANGHAI MAJORBIO BIO PHARM TECH
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