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A Noise Reduction Method for Targeted Capture Enrichment of Sequencing Library Molecules with Small Panel Probes

A targeted capture and sequencing library technology, applied in the field of biomedicine, can solve the problems of difficult to meet the requirements of sequencing depth and waste of sequencing cost

Active Publication Date: 2021-05-07
SHANGHAI ORIGIMED CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When using IDT's xGen Hybridization and Wash Kit for small panel probe hybridization capture, the library can be prepared faster and the operation is easier to implement. However, the off-machine sequencing data shows that the target rate needs to be improved, and the rRNA ratio is also high. It is a non-specific region, which makes it difficult to meet the requirements of sequencing depth under the premise of the same amount of data, resulting in waste of sequencing costs

Method used

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  • A Noise Reduction Method for Targeted Capture Enrichment of Sequencing Library Molecules with Small Panel Probes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Aiming at the problems in the current library preparation, the present invention uses the probe to hybridize with the library molecules, and after the streptavidin magnetic beads capture the library molecules in the target region, the preheated Stringent Wash Buffer is added to the streptavidin during the heating and washing process. and magnetic beads, and vortex for about 5 seconds, then transfer the mixture to a pre-warmed clean low DNA binding centrifuge tube and incubate for 5 minutes. At the end of the incubation, vortex the magnetic beads for about 5 seconds, centrifuge briefly, place on the magnetic stand, aspirate the supernatant, add preheated Stringent Wash Buffer again, and vortex for about 5 seconds, then transfer the mixture to the preheated clean Incubate the centrifuge tube for 5 minutes. Subsequent steps are consistent with the existing process. The heating and washing process is an important link in the library capture process, which directly determin...

Embodiment 2

[0073] Use IDT’s kit to construct a DNA library, wash with heat, and make sure that 1× Wash Buffer 1 is always preheated in a 70°C water bath. After the 30 min incubation of the sample mixture containing the library, probes, and streptavidin magnetic beads, remove the sample from the thermostat. Transfer 80 μL of pre-warmed 1× Wash Buffer 1 to the sample, and mix by pipetting 10 times. Transfer all the above Mix (sample mixture) to new 1.5mL LoBind Tubes, vortex for 5sec, and let the sample stand still. Discard the supernatant after the solution is completely clear. Remove the sample from the magnetic stand, transfer 100μL of preheated 1×Stringent Wash Buffer to the sample, vortex for 8sec, avoid excessive air bubbles, and transfer to new 1.5mL LoBind Tubes. After incubating the new LoBind Tubes on the metal bath for 5 minutes, vortex for 3 seconds, put the sample on the magnetic stand, and discard the supernatant after the solution is completely clear. Repeat the steps of a...

Embodiment 3

[0077] To construct an RNA library, the steps are basically the same as in Example 2. During heating and washing, make sure that 1.5mL LoBindTubes and 1×Stringent Wash Buffer are always preheated in a 67°C metal bath.

[0078] 3.1 After the sample mixture containing library, probe and streptavidin magnetic beads has been incubated for 45 minutes, take the sample out of the PCR machine.

[0079] 3.2 Transfer 100 μL of preheated 1× wash buffer 1 (metal bath) to the sample, and mix by pipetting 10 times to avoid excessive air bubbles.

[0080] 3.3 Transfer all the above sample mixture (Mix) to new 1.5mL LoBind Tubes, vortex for 5sec, place the sample on the magnetic stand, and discard the supernatant after the solution is completely clear.

[0081]3.4 Remove the sample from the magnetic stand, transfer 150μL of preheated 1×Stringent Wash Buffer to the sample, vortex for 5sec to avoid excessive air bubbles, and transfer to new 1.5mL LoBind Tubes.

[0082] 3.5 After incubating on...

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Abstract

The invention belongs to the field of biomedicine, and relates to a noise reduction method for targeted capture and enrichment of sequencing library molecules with small panel probes. The noise reduction method includes: acquiring library molecules, adding washing buffer to the mixture containing library, probe and streptavidin magnetic beads, removing non-specific hybridization by physical shaking, and removing the hybridization after decontamination. The product is moved to a new container and the rinsed library molecules are obtained. The noise reduction method for the probe of the present invention to target capture and enrich the sequencing library molecules, optimize the capture efficiency, reduce non-specific hybridization, improve the target rate, and can effectively reduce the rRNA ratio for the RNA library, and the present invention provides high-quality sequencing. library.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a library preparation method in an NGS detection process, in particular to a noise reduction method for targeted capture and enrichment of sequencing library molecules by small panel probes. Background technique [0002] High-Throughput Sequencing (High-Throughput Sequencing), also known as Next Generation Sequencing (NGS), is relative to traditional Sanger Sequencing (Sanger Sequencing). High-throughput sequencing can be divided into pyrosequencing, synthesis sequencing, ligation sequencing and ion semiconductor sequencing according to different principles and technologies. Representatives of major platforms include Roche's 454 sequencer (Roch GS FLX sequencer), Illumina's Solexa Genome Analyzer (Illumina Genome Analyzer) and ABI's SOLiD sequencer (ABI SOLiDsequencer). [0003] In pyrosequencing, the sequencing reaction is regulated by the release of pyrophosphate during the incorporati...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6806C12N15/10C40B50/06
CPCC12N15/1093C12Q1/6806C40B50/06C12Q2523/308C12Q2527/101C12Q2563/131C12Q2535/122
Inventor 王维锋郭明慧
Owner SHANGHAI ORIGIMED CO LTD
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