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Connector processing method for Small RNA next-generation sequencing and library building

A processing method and next-generation sequencing technology, which is applied in the field of DNA sequencing library construction, can solve problems such as ineffectiveness and limitation of small RNA sequencing analysis research, and achieve the effect of solving the incompatibility of sequencing reagents and facilitating sequencing analysis research

Active Publication Date: 2016-06-01
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 smallRNA needs special sequencing reagents to be sequenced, which greatly limits the sequencing analysis of smallRNA and its application in cancer diagnosis and follow-up. Applications in Targeted Therapy

Method used

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  • Connector processing method for Small RNA next-generation sequencing and library building
  • Connector processing method for Small RNA next-generation sequencing and library building

Examples

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Embodiment

[0031] In this example, smallRNA derived from the exosome in blood is used to construct a smallRNA 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 3’ adapter (RNA3’Adapter, abbreviated as RA3) to 5 μL of nuclease-free smallRNA aqueous solution, mix well, react in a Thermalcycler at 70°C for 2 min, and immediately place it on ice, namely reaction solution 1.

[0036] (2) Mix configuration

[0037] The Mix solution includes: 2 μL Ligation Buffer, 1 μL RNase Inhibitor, 1 μL T4RNALigase2 (Deletion Mutant), and mix well to make a Mix solution.

[0038] Add all the Mix solution to the reaction solution 1 placed on ice, react in the Thermalcycler at 28°C for 1 hour, add 1 μL StopSolution to the reac...

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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 SmallRNA 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 to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/10C40B50/06
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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