Sequencing method and kit for improving bar code splitting ratio

Abstract

The invention relates to a sequencing method and kit for improving the bar code splitting ratio. The method comprises the following steps of: providing an insert fragment sequencing primer, a blockedplaceholder primer and a template to be detected, wherein the 3' end of the placeholder primer is blocked and cannot extend; furthermore, the hybridization position of the placeholder primer on the template to be detected is located at the hybridization position of a bar code sequencing primer or a downstream region thereof; hybridizing the insert fragment sequencing primer and the placeholder primer onto the template to be detected, and sequencing an insert fragment; and, removing the placeholder primer and hybridizing with the bar code sequencing primer after sequencing of the insert fragment is completed and the 3' hydroxyl end of a sequencing product is blocked, or recovering the 3' end of the placeholder primer into a 3' hydroxyl group as the bar code sequencing primer to perform barcode sequencing. According to the method disclosed by the invention, on the premise of not influencing the quality of single-ended sequencing data, the problem that a part of sequences with too shortinsert fragments cannot be split due to the fact that the single-ended sequencing read length exceeds the length of the insert fragments is solved.

Description

technical field [0001] The invention relates to the technical field of sequencing, in particular to a sequencing method and a kit for improving the barcode splitting rate. Background technique [0002] Existing next-generation sequencing methods include insert sequencing and barcode sequencing. Inserts can be sequenced first, and then barcodes can be sequenced; or barcodes can be sequenced first, and then inserts can be measured. [0003] There is a problem with the method of sequencing inserts first, and then sequencing barcodes: the length of inserts in the library is diffusely distributed. When the inserts need to be read through, the adapters and barcodes of the shorter inserts will be detected. However, the sequencing primers that lead to the barcode cannot be combined with the sequence to be tested and cannot be sequenced, so that the part of the test to be tested that is too short in the inserted fragment cannot be split out and is filtered out. [0004] However, if ...

AI Technical Summary

Problems solved by technology

[0005] The present invention provides a sequencing method and a kit for improving the barcode splitting rate. Under the premise of not affecting the quality of the single-end sequencing data, it solves the problem that the read length of the single-end sequencing exceeds the length of the insert, which causes some of the inserts to be too short. Split out the problem

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