Barcode, linker sequence and kit for third-generation sequencing, and library construction method for third-generation sequencing

Abstract

The invention provides a barcode, a linker sequence and a kit for third-generation sequencing, and a library construction method for the third-generation sequencing. The barcode is composed of a plurality of consecutive bases, wherein in the bases, at least part of the bases are methylated bases. The linker sequence includes the above barcode. The methylated barcode used in the invention can splitthird-generation sequencing data which cannot be split by a conventional splitting method, so that the splitting rate of the third-generation sequencing data is greatly improved; and the overall splitting rate of the third-generation sequencing data can reach about 85%.

Description

technical field [0001] The invention relates to the technical field of third-generation sequencing, in particular to a tag sequence for third-generation sequencing, an adapter sequence, a kit, and a method for building a third-generation sequencing library. Background technique [0002] The third-generation sequencing (such as Pacbio platform sequencing) is based on the principle of sequencing-by-synthesis, using SMRT (single-molecule real-time fluorescent sequencing technology) chips as carriers for sequencing reactions. During sequencing, the genomic DNA is broken into many small fragments, and after being made into droplets, they are dispersed into different ZMW (zero-mode waveguides, zero-mode waveguides) nanopores. When the polymerization reaction at the bottom of the ZMW nanopore occurs, the nucleotides labeled with different fluorescence will be retained by the polymerase in the fluorescence detection area of ​​the small hole, and the type of base composition of the t...

AI Technical Summary

Problems solved by technology

When the library is long, the polymerase may not be able to read the tag sequence. At this time, most of the data cannot be split into each sub-library, resulting in data waste.

The insert length of the third-generation sequencing library is roughly 5-8kb, and the advantage of the third-generation sequencing read length is not fully utilized (currently, the read length is 15-20kb). At the same time, due to the problem of tag sequence design, the split rate is roughly 60 -70%, resulting in 30-40% data waste, which virtually increases the cost of sequencing and limits the development of the third-generation sequencing platform

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