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Method and kit for constructing sequencing library for simultaneously realizing genome copy number variation detection and gene mutation detection

A copy number variation and genome technology, which can be used in biochemical equipment and methods, chemical libraries, and microbial determination/inspection. It can solve the problems of high sequencing cost, inability to detect three variants, and limited sample size.

Pending Publication Date: 2020-07-07
BERRYGENOMICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For general samples, this can be achieved by building the library twice and using the computer. The disadvantages are cumbersome operation, long cycle and low efficiency.
However, for some cell samples (e.g. rare cells), due to limited sample size, it is often not possible to build a library twice, so it is not possible to detect all three variants
Even with whole-genome deep sequencing of rare cell samples, the very high cost of sequencing prevents large-scale application

Method used

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  • Method and kit for constructing sequencing library for simultaneously realizing genome copy number variation detection and gene mutation detection
  • Method and kit for constructing sequencing library for simultaneously realizing genome copy number variation detection and gene mutation detection
  • Method and kit for constructing sequencing library for simultaneously realizing genome copy number variation detection and gene mutation detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1. Construction of a DNA sequencing library according to the method of the present invention

[0048] Step 1: Lyse white blood cells (blood samples from healthy people) and extract DNA. Reaction mixtures as shown in Table 1 were prepared, incubated at 60°C for 20 minutes, 95°C for 4 minutes, and then the samples were kept at 4°C.

[0049] Table 1:

[0050]

[0051] Step 2: Pre-amplification of genomic DNA. The pre-amplification primers used in this step include random primers (sequence: 5'GCTCTTCCGATCTKKKKKKKKKKN*N*N 3', wherein K represents G or T bases, and N represents random degenerate bases A / T / C / G , *represents sulfomodification) and specific primers, wherein the specific primers are designed for exon 2 and exon 3 of the HBB gene (NCBI ID: NM_000518, which is known to cause β-thalassemia due to its mutation), which The sequences are shown in Table 2 below (the underlined GCTCTTCCGATCT represents the general region in the specific primers).

[0052] ...

Embodiment 2

[0063] Example 2. Detecting the quality of the DNA sequencing library

[0064] The DNA sequencing library prepared in Example 1 was purified, and then the concentration was detected by Qubit. The concentration of the blank control should be <10ng / μl, and the concentration of the sample should be ≥10ng / μl, and the samples whose concentration meets the requirements should be quantified by qPCR. According to the quantitative results of qPCR, the library was subjected to 36 bp single-end sequencing in accordance with the standard operating procedures of the sequencer. Compare the single-end sequencing results with the human genome reference sequence, detect the genome copy number variation of each sample, and analyze the library quality and HBB amplification results.

[0065] figure 1 The Manhattan plot of the high-throughput sequencing results of the three samples prepared according to Example 1 is shown. Such as figure 1As shown, the sequencing results of the three samples a...

Embodiment 3

[0070] Example 3. Effect of the ratio of random primers to specific primers on the quality of the sequencing library

[0071] The DNA sequencing library was prepared according to the method of Example 1, the difference being that the composition and content of the pre-amplification primers are shown in Table 6 below:

[0072] Table 6

[0073]

[0074] The prepared DNA sequencing library was purified, and then the concentration was detected by Qubit. The concentration of the blank control should be <10ng / μl, and the concentration of the sample should be ≥10ng / μl, and the samples whose concentration meets the requirements should be quantified by qPCR. According to the quantitative results of qPCR, the library was subjected to 36 bp single-end sequencing in accordance with the standard operating procedures of the sequencer. Compare the single-end sequencing results with the human genome reference sequence, detect the genome copy number variation of each sample, and analyze t...

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Abstract

The invention relates to a method and a kit for constructing a DNA sequencing library. More specifically, the invention provides a method for constructing a DNA sequencing library for simultaneously realizing single-cell genome copy number variation detection and gene mutation detection. The method is characterized by comprising the following steps: 1) cracking cells to release gene group DNA; 2)carrying out pre-amplification on the genome DNA by using a mixed primer consisting of a random primer and a specific primer; and 3) carrying out secondary amplification on the pre-amplified genome DNA to obtain the DNA sequencing library. The invention also relates to a method and a kit for simultaneously realizing single-cell genome copy number variation detection and gene mutation detection.

Description

technical field [0001] The present invention relates to methods and kits for constructing DNA sequencing libraries. More specifically, the present invention relates to a method and a kit for constructing a DNA sequencing library capable of simultaneously realizing single-cell genome copy number variation detection and gene mutation detection. Background technique [0002] With the advancement of science and technology, traditional Sanger sequencing can no longer fully meet the needs of research. Genome sequencing requires lower cost, higher throughput, and faster sequencing technology. High-throughput sequencing (also known as second-generation sequencing) ) technology came into being. The core idea of ​​high-throughput sequencing technology is sequencing-by-synthesis, that is, to determine the DNA sequence by capturing the markers of newly synthesized ends. Existing technology platforms mainly include Roche / 454FLX, Illumina / Hiseq, Miseq, NextSeq and Life Technologies / SOL...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12Q1/6806
CPCC40B50/06C12Q1/6806C12Q2531/113
Inventor 贾哲陈迪张建光
Owner BERRYGENOMICS CO LTD
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