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A linker processing method for small RNA next-generation sequencing library construction

A processing method and second-generation sequencing technology, which are applied in the field of DNA sequencing library construction, can solve the problems of limiting small RNA sequencing analysis and research, low yield, etc., and achieve the effect of facilitating sequencing analysis and research and solving the incompatibility of sequencing reagents.

Active Publication Date: 2020-03-24
北京六合华大基因科技有限公司 +1
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Problems solved by technology

[0003] Since the 20th century, with the advancement of modern medical technology in the diagnosis and treatment of cancer, the cancer mortality rate has declined, but with little effect
When performing sequencing, the sequencing reagents of the PEI library and the SE library are different. The SE library of small RNA needs to use special sequencing reagents for sequencing, which greatly limits the sequencing analysis of small RNA and its application in cancer diagnosis. and subsequent application in targeted therapy

Method used

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  • A linker processing method for small RNA next-generation sequencing library construction
  • A linker processing method for small RNA next-generation sequencing library construction

Examples

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Embodiment

[0031] In this example, small RNA derived from the exosome in blood is used to construct a small RNA library. The library construction in this example includes adapter processing, library PCR, and recovery of PCR amplification products.

[0032] Among them, the joint processing method is as follows:

[0033] 1. Small RNA fragments are connected to the 3' adapter of the SE library

[0034](1) Reaction solution 1

[0035] Add 1 μL of the 3' adapter (RNA 3' Adapter, RA3 for short) to 5 μL of nuclease-free small RNA aqueous solution, mix well, react in a Thermal cycler at 70°C for 2 minutes, and immediately place it on ice, namely reaction solution 1 .

[0036] (2) Mix configuration

[0037] Mix solution includes: 2 μL Ligation Buffer, 1 μL RNase Inhibitor, 1 μL T4 RNA Ligase 2 (Deletion Mutant), mix well to make Mix solution.

[0038] Add all the Mix solution to the reaction solution 1 placed on ice, react in the Thermal cycler at 28°C for 1 hour, add 1 μL Stop Solution to th...

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Abstract

The application discloses a connector processing method for Small RNA next-generation sequencing and library building and application of the connector processing method. According to the application, the connector processing method comprises the following steps: A, adding SE library 3' connector and 5' connector to two ends of a Small RNA segment; B, conducting reverse transcription on the Small RNA segment that the connectors are linked, so as to obtain cDNA; C, adding 'A' to the 3' end of the cDNA segment obtained from Small RNA segment; and D, connecting a PEI 'Y'-shaped connector on the cDNA segment obtained in the step C. According to the connector processing method of the application, by adding the PEI connector on the basis of the SE connector, the built Small RNA library can make use of a PEI sequencing reagent during sequencing, so as to solve the problem on the compatibility of the SE library and the PEI library sequencing reagent and to facilitate Small RNA sequencing; therefore, the connector processing method can offer convenience for cancer diagnosis and subsequent targeted therapy based on the Small RNA sequencing.

Description

technical field [0001] This application relates to the field of DNA sequencing library construction, in particular to a linker processing method applied to Small RNA next-generation sequencing library construction and its application. Background technique [0002] The latest "Global Cancer Report 2014" released by the World Health Organization pointed out that in 2012, the number of cancer patients and deaths in the world increased, and the number of new cases and deaths in China ranked first in the world. According to the "2012 China Cancer Registration Annual Report" released by the my country Cancer Registration Center, there are estimated to be about 3.12 million new cancer cases in the country every year, with an average of 8,550 people per day, and 6 people are diagnosed with malignant tumors every minute in the country. In the national age group of 35 to 39 years old, an average of 87.07 people per 100,000 people will suffer from cancer, while in the age group of 40 t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6806C12Q1/6869C12N15/11C12N15/10C40B50/06
Inventor 赵鑫周鑫兰侯勇吴逵
Owner 北京六合华大基因科技有限公司
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