A method for constructing a sequencing library, reagents for building a library, and applications thereof

Abstract

The application discloses a method for constructing a sequencing library, a reagent for building a library and an application thereof. The sequencing library construction method of the present application includes using degenerate primers to perform constant temperature amplification on the enriched target gene, and then performing end repair, adding "A" bases, and adapters to the constant temperature amplification products, and purifying to obtain a sequencing library; And the 5' end of the primer has a tag sequence, and the 3' end is a random sequence. The sequencing library construction method of the present application uses degenerate primers to amplify the target gene at a constant temperature to obtain fragmented DNA suitable for sequencing, which omits the fragmentation step and avoids the loss of tag sequences and useless sequencing data. The sequencing library construction method of the present application simplifies the library construction process, shortens the library construction cycle, reduces the cost of library construction, improves data utilization, and provides a new library construction scheme for high-throughput sequencing.

Description

technical field [0001] The present application relates to the field of sequencing library construction, in particular to a sequencing library construction method, library construction reagents and applications thereof. Background technique [0002] Thalassemia is caused by a gene defect. One or more globin synthesis abnormalities in hemoglobin lead to anemia or a pathological state. There are various types of gene mutations, including multiple deletions and point mutations. Thalassemia detection methods include Sanger sequencing, qPCR, NGS, probe capture sequencing, gene chip typing, etc. Using high-throughput sequencing to detect thalassemia mutations has the characteristics of low cost, high throughput, and high accuracy. [0003] In the prior art, the method for constructing a thalassemia library based on the Illumina Hiseq2500 sequencing platform mainly includes the following steps: [0004] (1) using specific primers with specific tag sequences to amplify the human he...

AI Technical Summary

Problems solved by technology

[0011] The above method of sequencing library construction or the thalassemia detection method based on this library has its own limitations, for example: first, the final output data in the above method must have both library tags and specific primer tags to distinguish sample information, However, some DNA fragments lose specific primer tags due to interruption and become useless data, resulting in data waste and increased sequencing costs; the library tags are included in the specific adapters of the above step (5), which is to perform specificity on the source of the library. A nucleic acid sequence of 6-10bp identified; the specific primer label is the specific label sequence carried by the specific primer in step (1), which is a section of nucleotides that specifically identifies the target fragment amplified by the specific primer Acid sequence, its length can be adjusted according to the specific conditions of the primer

Second, the above method uses PE sequencing for fragment completion, so as to achieve full coverage of the gene sequence; the fragment selection range is large, and the steps of gel cutting to select fragments are cumbersome

Third, the library construction period is long, and the multi-step purification causes the loss of the DNA library and increases the cost of library construction

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