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A method for constructing a tandem rad tag sequencing library

A construction method and tag sequencing technology, applied in the field of molecular biology DNA genetic markers and DNA methylation detection, can solve the problem that the number of tandem tags cannot be effectively controlled, the tag connection sequence, the tag length is short, and the paired-end long read length cannot be used. Sequencing and other problems, to achieve the effect of reducing the cost of library construction and sequencing, reducing the cost of library construction, flexible screening and detection

Active Publication Date: 2017-04-26
OCEAN UNIV OF CHINA
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AI Technical Summary

Problems solved by technology

The limitation of the existing 2b-RAD or MethylRAD technology is that the tag length generated by the library construction is short (~35bp), which can only be used for single-end 35-50bp sequencing, and cannot be applied to more cost-effective Advantageous paired-end long-read sequencing (such as PE100-150bp sequencing)
[0004] In addition, the serial analysis of gene expression (SAGE), which is applied in the field of gene expression analysis, connects the representative tags of transcripts to form concatemers of different lengths, but this technology cannot effectively control the number of tandem tags. The number and connection order of the tags, and the analysis method of the tandem DNA sequence is also cloned into a plasmid vector for sequencing analysis. It has not proposed a sequencing library construction plan for sequentially concatenating more than three tags on the next-generation sequencing platform, and the sequencing library Simultaneously realize SNP typing and methylation detection

Method used

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  • A method for constructing a tandem rad tag sequencing library
  • A method for constructing a tandem rad tag sequencing library
  • A method for constructing a tandem rad tag sequencing library

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Embodiment 1

[0083] The following uses the scallop as the experimental material, and tandem sequencing of different types of tag libraries as an example to describe the library construction method of this embodiment in detail. For the reagents and reaction conditions used in this embodiment, those skilled in the art can follow this embodiment. The technical solution of the example is selected in the prior art, and is not limited to the limitation of the specific example of this example.

[0084] 1. Extraction of scallop genome DNA

[0085] Get about 0.1 g of the adductor muscle of a scallop and add it to 500 μL STE lysis buffer. The STE lysis buffer includes NaCl: 100 mmol / L; EDTA: 1 mmol / L, pH=8.0; Tris-HCl, 10 nmol / L, pH=8.0, cut into pieces, then added 50 μL of 10% SDS (sodium dodecyl sulfate), and 5 μL of proteinase K (20 mg / mL), digested in a water bath at 56 ° C until the tissue fragments were completely lysed, and the lysate clarify. Add an equal volume of saturated phenol (250 μ...

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Abstract

The invention discloses a method for constructing a tandem RAD tag sequencing library. The steps are: 1) Digestion: use an endonuclease to perform enzyme digestion reaction on DNA; The restriction site of SapI enzyme, the characteristic sequence used to achieve tag tandem and the universal sequence for amplification primer binding; 3) Amplification of the ligation product: PCR amplification, enrichment, and gel cutting using a combination of biotin primers and common primers Recover the PCR products, re-amplify, mix and purify in equal amounts; 4) Tandem tag library: Use SapI enzyme to digest the PCR products, and serialize them sequentially; 5) Tandem long tag enrichment: Gel purify the tandem long tags and use primers Perform PCR amplification and introduce barcode to construct a library; 6) Library sequencing The present invention can realize high-throughput and low-cost screening and detection of genome-wide genetic markers and epigenetic variations.

Description

technical field [0001] The invention belongs to the technical field of molecular biology DNA genetic markers and DNA methylation detection, and in particular relates to a method for constructing a tandem RAD tag sequencing library. Background technique [0002] In recent years, the rapid development of high-throughput sequencing technology has greatly promoted the depth and breadth of animal and plant genomics research. Reduced genome technology is a genome sequencing analysis technology that uses restriction endonucleases to reduce genome complexity. Because it uses the sequence corresponding to the enzyme-cut fragment of a certain size as a partial representative of the entire genome sequence, it reduces the complexity of the genome and is low in cost, independent of reference genome information. These advantages make non-model organisms that are relatively scarce in genome information It is possible to carry out omics analysis, which has been widely used in genetic map c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B50/06C12Q1/68
CPCC12N15/1065C12Q1/6869C12Q2525/191C12Q2563/185C12Q2521/301C12Q1/686C12Q2563/179C12Q1/68C40B50/06C12N15/1068
Inventor 王师包振民刘平平吕佳张玲玲
Owner OCEAN UNIV OF CHINA
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