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

An immune library and full-length technology, applied in the field of gene sequencing, can solve problems such as the lack of key variable region sequences, achieve excellent amplification and enrichment effects, simplify experimental operations, and simplify experimental procedures

Active Publication Date: 2020-09-15
WUHAN FRASERGEN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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.

Method used

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

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: the synthesis of primer

[0049] The primer sequences designed in the present invention can be synthesized in various primer synthesis companies, such as Shanghai Sangong, and the primer sequences provided are shown in Table 1.

[0050] Table 1: Sequences of designed primers

[0051]

[0052]

[0053]

[0054] Among them, BCR Template Switching Oligo primers and 5'-end universal primers are designed based on the consistent sequence of the constant regions of different types of BCR immune repertoires; 3'-end specific primers are designed based on conserved regions, covering the entire BCR immune repertoire The full-length sequence of the library.

[0055] In addition, V in the Oligo dT primer is a degenerate base, representing one of G, A, and C; in the BCR TemplateSwitching Oligo primer, [dN(12)] is represented as a 12-base UMI tag sequence, composed of 12 random bases The base composition is used to correct the bias of the amplification, that i...

Embodiment 2

[0059] Example 2: Synthesis of cDNA first strand in total RNA

[0060] The primers used in the experimental steps were synthesized in Shanghai Sangong, and the primers were uniformly diluted to 10uM. The first step of the project embodiment of cDNA first-strand synthesis is as follows:

[0061] 1) Oligo dT reverse transcription primer combined with poly(A)

[0062] Table 2:

[0063]

[0064] Gently flick to mix, centrifuge briefly, incubate at 70°C for 5 minutes and place on ice immediately.

[0065] 2) Reverse transcription to synthesize the first strand of cDNA

[0066] Prepare the following reaction:

[0067] table 3:

[0068]

[0069] Gently flick to mix, centrifuge briefly, and incubate at 42°C for 75min. Immediately after the completion of the reaction, place it on ice, add 1uL of BCRTemplate Switching Oligo, flick to mix well, centrifuge briefly, and incubate at 42°C for 15min.

Embodiment 3

[0070] Example 3: Full-length amplification of BCR cDNA

[0071] The full-length amplification of BCR cDNA includes two rounds of semi-nested amplification reactions. The first round of amplification carries out the preliminary enrichment of BCR sequences, and the band uniformity of the amplified sequences is poor. The second round of amplification uses internal Nested primers further improve the specificity of amplification and make the amplified band single.

[0072] 1) The first round of PCR amplification of the full length of BCR cDNA

[0073] Take a new 0.2mL PCR tube and add the following reagents:

[0074] Table 4:

[0075]

[0076]

[0077] Mix well, centrifuge briefly, and place on a PCR instrument for PCR reaction: incubate at 98°C for 2 minutes; incubate at 98°C for 20 s, at 65°C for 15 s, and at 72°C for 45 s, a total of 18 cycles; then incubate at 72°C for 5 min.

[0078] After the reaction, it can be stored in a -20°C refrigerator for a long time.

[00...

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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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Claims

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Application Information

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Owner WUHAN FRASERGEN CO LTD
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