Three-generation library constructing and sequencing method for overall length amplification of BCR immunity group library

Abstract

The invention discloses a three-generation library constructing and sequencing method for overall length amplification of a BCR immunity group library. The method comprises the steps of (1) designingand synthesizing primers according to the constant region of a BCR consistency sequence; (2) synthesizing a first chain cDNA under the action of an Oligo dT primer and a BCR Template Switching Oligo primer; (3) performing first-round amplification and second-round amplification on the overall length of BCR cDNA; (4) mixing samples of BCR overall length amplicon fragments; (5) performing library construction; and (6) performing three-generation sequencing. According to the method disclosed by the invention, multiple primers are designed in the conservative region of a BCR constant region, so that the overall length sequences of the whole BCR are covered. During BCR sequence amplification, in order to further improve the validity of 3' end amplification, multiple groups of semi-nest primersare designed, and through matching and screening of the semi-nest primers, amplicon fragments covering the whole BCR overall length can be obtained. Through a PacBio library constructing technologicalprocess, sequencing contacts having barcode are added to the two ends of each amplicon, and then PacBio sequencing can be performed; and through splitting and data correction, HiFi Reads of which theaccuracy rate is 99% or above can be obtained.

Description

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AI Technical Summary

Benefits of technology

The technical effects described in this patented technology include improved methods for analyzing DNA samples such as blood cells (blood), bone marrow aspirates, urine sediments, tissue biopsies, cell lines from cancer patients, etc., while reducing complexity and cost associated therewith. By performing these techniques at once, it becomes possible to analyze multiple targets simultaneously within short periods of time.

Problems solved by technology

Technological Problem addressed in this patented technical solution describes how to accurately identify unknown DNA fragments within certain parts called genome units, particularly when they are shorter than usual due to their ability to bind specifically to proteins involved in development processes like autoimmunity responses. Current techniques involve analyzing these shortened portions individually from multiple copies of mature BCR elements. However, current sequencers have limitations in terms of resolution and sensitivity, leading to incomplete coverage across partial domains. To address this issue, new technologies involving deep sequential analysis were developed based upon advanced sequencing tools.

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