Method for building sequencing library by hybridization

Abstract

Disclosed are a group of isolated nucleic acid tags for constructing a genome sequencing library, oligonucleotides, blocking sequences, a kit, a method for constructing a genome sequencing library, the genome sequencing library constructed, a method for sequencing a particular region in the genome of a sample, and a method for sequencing the genomes of a plurality of samples. The group of isolated nucleic acid tags is comprised of nucleotides as set forth in SEQ ID NO: (165+M), where M = any integer between 1-159.

Description

technical field [0001] The present invention relates to the technical field of nucleic acid sequencing, in particular to the field of sequence capture technology, especially the sequence capture technology based on hybridization. In addition, the present invention also relates to labeling technology and a method for realizing sequence capture of multiple samples in the same reaction system. The method of the present invention is particularly suitable for the second generation sequencing technology, especially the solexa sequencing technology. The methods of the invention are useful in genomics research. Background technique [0002] The second-generation sequencing technology represented by Illumina solexa, AB Solid and Roche 454 has greatly reduced the cost of sequencing, has developed rapidly in recent years, and has become an important tool for genomics research. Compared with the sanger sequencing technology of the chain termination method, the second generation sequen...

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Problems solved by technology

[0010] 1. The method of introducing tag sequences through adapters is not conducive to the application of this technology in sequencing platforms such as Solexa: on the one hand, the tag sequences added after adapter ligation are located between the sequencing primers and the sample DNA, and 11 bp must be sequenced before sequencing the sample DNA The method of sequentially sequencing the tag sequence and sample DNA with the same sequencing primer is used in the second-generation sequencing technology platform with a relatively short sequencing length, which will undoubtedly further shorten the effective sequencing length of the sample DNA; On the one hand, the introduction of tag sequences based on adapter ligation will cause both ends of the sample DNA to carry tag sequences, which will cause the tag sequences to be sequenced twice when sequencing platforms such as Solexa perform paired-end sequencing, resulting in a waste of sequencing data

[0011] 2. This technology does not use adapter blocks (also called blocking sequences), which will reduce the binding efficiency of the sample DNA and the probe due to the annealing between the adapter complementary sequences during the hybridization of the sample, affecting the sequence capture effect. At the same time, the sample DNA without any association may be connected due to the annealing between the adapters, and cascade amplification to form "macromolecular DNA". The target DNA is captured together, resulting in a large number of non-target sequences in the capture sequence

[0012] 3. Since this technology is mainly optimized for chip hybridization, when it is used in a liquid phase system, due to the small initial amount of sample DNA, they may have a greater impact on the sequence capture effect after they are connected together through adapters. Potentially captures a large number of off-target sequences

Therefore, the sequence capture effect of this technical solution on the Agilent liquid phase hybridization platform may be poor

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