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A high-throughput simplified genome sequencing library construction method

A genome sequencing and construction method technology, applied in chemical libraries, combinatorial chemistry, recombinant DNA technology, etc., can solve the problems of large amount of genomic DNA, discarding original data, poor sequencing quality, etc., to achieve high throughput, reduce impact, and cost. low effect

Active Publication Date: 2017-10-10
BIOMARKER TECH
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

At present, the next-generation sequencing library construction methods that can be used for population genetics research mainly include whole genome resequencing (WGR, Whole genome resequencing), RAD-Seq (Restriction-site-association DNA sequencing), GBS (Genotyping-by-Sequencing), etc. However, these methods have different problems. For example, WGR needs a high-quality reference genome sequence and the cost of building a library is high; although RAD-seq can be used for species without a reference genome, the amount of genomic DNA is large and the process is complicated. And the sequencing quality is poor, nearly half of the original data will be discarded due to sequencing errors; although the steps of GBS are relatively simple, it can only collect short fragments and the number of fragments is relatively small

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  • A high-throughput simplified genome sequencing library construction method
  • A high-throughput simplified genome sequencing library construction method
  • A high-throughput simplified genome sequencing library construction method

Examples

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Embodiment 1

[0043] Example 1 High-throughput Simplified Genome Sequencing Library Construction of the Genetic Map of Mei F1 Population

[0044] 387 offspring of the Prunus mune F1 population and their two parents were selected as experimental materials, and the construction of a high-throughput simplified genome sequencing library included the following steps:

[0045] Step 1: Genomic DNA of 387 progeny of Mei F1 population and its two parents was diluted to 50ng / μL for later use; genomic DNA fragmentation: the diluted genomic DNA was digested with blunt end restriction endonucleases HaeIII and Hpy166II, The enzyme digestion reaction system is:

[0046]

[0047] The mixed system was incubated in a constant temperature water bath at 37°C for 5 hours.

[0048] Step 2: Add the reaction reagents required to generate the sticky A-terminus at the 3' end to the digestion reaction product of the first step, including:

[0049]

[0050] The mixed system was incubated in a constant temperatur...

Embodiment 2

[0090] Example 2 Construction of High-throughput Simplified Genome Sequencing Library of Flax Genetic Map

[0091] In this embodiment, 115 offspring of the flax F2 population and their two parents were selected as experimental materials, and the genomic DNA was diluted according to the first step in Example 1; the genomic DNA fragment was double-digested with RsaI and HaeIII, and the reaction system was the same as in Example 1; Add A to the 3' end according to step 2 in Example 1; connect each sample with a barcode adapter according to step 3 in Example 1; perform PCR reaction according to step 4 in Example 1 (primers for PCR reaction and conditions are the same as in Example 1); the PCR product is purified and detected; the PCR purified products of 2 parents and 115 progeny are mixed according to a mass ratio of 40:8 and cut into gel to select fragments, and a fragment of 450-550bp is selected; according to the embodiment In step 5 of 1, perform low-cycle PCR amplification a...

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Abstract

The invention provides a method for constructing a high-flux simplified genome sequencing library. The method comprises the following steps: digesting genome DNA of different samples by using restriction enzymes, thereby obtaining blunt-end DNA fragments with phosphorylation modification at the 5' end; adding the base A at the 3' end, connecting the DNA fragments of different samples with 5' phosphorylation and 3' cohesive ends A with joints containing different bar code sequences, connecting the products PCR, purifying and mixing the amplification products, performing low-cycle PCR amplification, thereby obtaining a target fragment amplification product of which the ratio of the joints to joint dimers is greatly reduced; and separating, purifying, and forming the high-flux simplified genome sequencing library from the purified products. The method disclosed by the invention has the advantages that the flux is high, the cost is low, and the generated library is high in sequencing quality and has very wide application prospects in researches such as high-flux SNP detection and marker development, genetic map drawing and functional gene location.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a high-throughput simplified genome sequencing library. Background technique [0002] With the development and upgrading of next-generation sequencing technology, especially the Illumina sequencing platform, the research of population genetics has undergone revolutionary changes. Using next-generation sequencing, hundreds of thousands or even millions of SNPs information can be obtained, and more and more Multiple economically important animals and economic crops were located in candidate areas closely associated with important economic traits. At present, the next-generation sequencing library construction methods that can be used for population genetics research mainly include whole genome resequencing (WGR, Whole genome resequencing), RAD-Seq (Restriction-site-association DNA sequencing), GBS (Genotyping-by-Sequencing), etc. However, these methods have d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/10C40B50/06
Inventor 郑洪坤刘慧
Owner BIOMARKER TECH
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