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A Hybridization Capture Method for Genome Target Region Sequencing

A technology of target region capture and hybridization capture, which is applied in the field of hybridization capture for genomic target region sequencing, can solve the problems affecting the sequencing process, different operating conditions, and undisclosed capture probes, so as to improve capture efficiency, moderate incubation time, The effect of reducing the cost of capture

Active Publication Date: 2021-03-23
北京益序医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, prolonged incubation significantly affects the sequencing process.
In addition, higher incubation temperature and longer incubation time will affect the concentration of salt ions in the mixture, especially when the volume of the hybridization reaction is small
[0005] Although many sequencing companies have been able to provide commercial hybridization enrichment methods and corresponding kits, their hybridization methods are mostly aimed at the probes produced by the company, and the capture probes are usually not disclosed, and the reagent solution is expensive
The operating conditions of each kit are different, and there are large differences, and the input amount of the library has strict requirements

Method used

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  • A Hybridization Capture Method for Genome Target Region Sequencing
  • A Hybridization Capture Method for Genome Target Region Sequencing
  • A Hybridization Capture Method for Genome Target Region Sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1 reagent preparation and storage

[0050] The water used for the preparation of the following reagents is nucleic acid-free water, and the containers used must be treated with nuclease before use.

[0051] 1) Note #1

[0052] 4*SSPE (ivitrogen, 15591043), packed in 1.5ml tubes, stored at 4 degrees.

[0053] 2) Note #2

[0054] 0.1M EDTA, pH 8.0 (ivitrogen, 15575020), aliquoted in 1.5ml tubes, stored at 4 degrees.

[0055] 3) Note#3

[0056] 50*denharts (invitrogen, 750018), packed in 1.5ml tubes, stored at -20 degrees.

[0057] 4) Note #4

[0058] 0.1% SDS (invitrogen, 15553027), diluted to 1 ×, distributed in 1.5ml tubes, stored at 4 degrees.

[0059] 5) Binding buffer:

[0060] Composition: NaCl, Tris-HCl, EDTA, pH7.5

[0061] Preparation:

[0062]

[0063] 6) Wash Buffer 1 (Wash Buffer 1):

[0064]Composition: SSC, SDS

[0065] Preparation:

[0066]

[0067] 7) Wash Buffer 2 (Wash Buffer 2):

[0068] Composition: SSC, SDS

[0069] Prep...

Embodiment 2

[0079] Example 2 Design and customization of panel

[0080] 1. Generation of target area list

[0081] a) According to the design of the experiment, select the region to be sequenced, and use hg19 as the reference genome to establish a list of target regions, including chromosome number and starting point location;

[0082] b) Target region adjustment: If the target region is less than 220bp, extend to both ends to 220bp.

[0083] 2. Assess the GC content of the target region

[0084] For regions with a GC content higher than 70% or lower than 30%, design 1× to 2× tiling probes.

[0085] 3. Probe Generation

[0086] a) Generate a probe group with a length of 110bp in the form of a tile array (tiling array), that is, regularly intercept the sequence of the genome according to a certain degree of overlap as the probe sequence (such as 2 × tiling design, the interval length is 55bp; 3 ×tiling design, interval length is 37bp);

[0087] b) For regions with a GC content higher ...

Embodiment 3

[0100] The preparation of embodiment 3 probe

[0101] This example introduces the preparation and storage of probes. The consumables required for the experiments in this chapter must be non-nucleic acid consumables. Gloves and masks should be worn throughout the process, and the operation should be performed on a clean bench.

[0102] 1. Probe template preparation

[0103] 1) First take 5μl of the probe stock solution, dilute it to 3ng / μl with 10mM Tris-HCl buffer, aliquot 4.2μl / tube, take one tube and continue with the following steps, and freeze the rest at -80°C;

[0104] 2) According to the following system and procedures, do 2 tubes in parallel;

[0105] reaction system:

[0106] KAPA HIFI library amplification Kit 25μl Rev (25μM) 1μl SP6promoter (25μM) 1μl Oligo pool (after dilution) 1μl water 22μl

[0107] Reaction procedure:

[0108]

[0109] 3) Qubit quantification: Take 1 μl of PCR product for qubit quantification, ...

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Abstract

The invention provides a genome object region sequencing hybrid capture method. The method comprises the steps of primer design and synthesis, library construction and hybrid capture, hybrid capture is optimized and is easy to operate and moderate in incubating time and can be used for various DNA libraries with different sources, the capture cost is effectively lowered, the capture efficiency is improved, the experimental time is shortened, and the method is suitable for genome object region sequencing.

Description

technical field [0001] The invention relates to the field of gene sequencing, in particular to a hybrid capture method for sequencing a genome target region. Background technique [0002] In next-generation sequencing, it is often necessary to sequence specific genomic target regions. For target region sequencing, the more commonly used method uses RNA probes to capture DNA fragments that enrich the target gene or genomic region of interest to improve the depth, accuracy and sensitivity of sequencing. [0003] Since DNA capture probes are designed to be complementary to the target region, the enrichment of target fragments depends on the physical and chemical properties of nucleotides, such as secondary structure, annealing temperature, nucleotide concentration, and GC content. and salt concentration. [0004] Based on the principle of hybridization enrichment, the specificity is usually improved by increasing the incubation time, and the usual incubation time is 10-72 hou...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6869
CPCC12Q1/6816C12Q1/6869C12Q2527/125
Inventor 张彦伟
Owner 北京益序医疗科技有限公司