Method for constructing series connection RAD [restriction-site-associated DNA (deoxyribonucleic acid)] tag sequencing libraries

Abstract

The invention discloses a method for constructing series connection RAD [restriction-site-associated DNA (deoxyribonucleic acid)] tag sequencing libraries. The method includes steps of 1), carrying out enzyme digestion reaction on DNA by the aid of incision enzymes; 2), respectively connecting linkers to enzyme digestion fragments; 3), amplifying connection products, to be more specific, carrying out PCR (polymerase chain reaction) amplification by the aid of biotin primer and common primer combinations, enriching PCR products, recycling the PCR products by means of gel incision, amplifying the PCR products again, equivalently mixing the PCR products with one another and equivalently purifying the PCR products; 4), serially connecting tag libraries with one another, to be more specific, carrying out enzyme digestion on the PCR products by the aid of SapI enzymes and sequentially serially connecting the PCR products with one another; 5) enriching series connection long tags, to be more specific, purifying the series connection long tags by the aid of gel, then carrying out PCR amplification by the aid of primers and introducing barcode to construct the libraries; 6), carrying out library sequencing. The linkers are provided with enzyme digestion sites of the SapI enzymes, signature sequences for serially connecting the tags with one another and universal sequences for binding the amplification primers. The method has the advantage that genetic markers and epigenetic variation can be screened and detected in whole genome ranges by the aid of the method in a high-throughput and low-cost manner.

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...

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

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