Library construction and sequencing method of small RNA (Ribonucleic Acid)

A library construction and library technology, applied in the field of small RNA library construction method and sequencing

Inactive Publication Date: 2018-05-22
SHENZHEN HUADA GENE INST
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Problems solved by technology

At present, there are no related studies and reports on small RNA CG library construction and small RNA CG sequencing; therefore, to study a library constructi

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  • Library construction and sequencing method of small RNA (Ribonucleic Acid)

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Embodiment

[0068] This example provides a new small RNA library construction and sequencing method, which can be used for the construction of small RNA libraries from all sources. The constructed small RNA CG library is suitable for the second-generation sequencing technology Complete Genomics (CG) sequencing platform.

[0069] The library construction method of the small RNA CG library in this example, such as figure 1 shown, including the following steps;

[0070] (1) Ligate the small RNA fragment to the 3' adapter of the CG library;

[0071] (2) connecting the product of step (1) to the 5' adapter of the CG library;

[0072] (3) Reverse transcribing the product of step (2) into cDNA;

[0073] (4) performing PCR amplification on the cDNA obtained in step (3), and purifying and recovering the PCR amplification product;

[0074] (5) performing single-strand separation on the purified and recovered PCR amplification product, and circularizing the obtained single-stranded DNA;

[0075...

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Abstract

The invention discloses a library construction and sequencing method of small RNA (Ribonucleic Acid). The construction method comprises the following steps: (1) connecting a small RNA segment with a 3' connector of a CG library; (2) connecting a product of step (1) with a 5' connector of the CG library; (3) carrying out reverse transcription on a product of step (2) to form cDNA (complementary Deoxyribonucleic Acid); (4) carrying out PCR (Polymerase Chain Reaction) amplification on the cDNA; purifying and recycling a PCR amplification product; (5) carrying out single-chain separation on the purified and recycled PCR amplification product and cyclizing single-chain DNA; (6) carrying out enzyme digestion on a cyclized product and recycling to obtain a small RNA CG library. According to the construction method disclosed by the invention, a novel CG library construction scheme is provided for the small RNA so that whole-genome level small RNA sequencing based on CG sequencing is realized and a foundation is laid for novel small RNA molecule digging, cancer target gene predication and identification, difference expression and analysis between samples, small RNA clustering, expression pattern analysis and the like and scientific evidence is also provided for cancer early diagnosis and targeting treatment and detection.

Description

technical field [0001] This application relates to the field of small RNA library construction and sequencing, in particular to a small RNA library construction method and sequencing method. Background technique [0002] According to epidemiological data in recent years, there are 600,000 new cases of lung cancer in my country, accounting for 19.59% of new cases of malignant tumors in that year, ranking first in malignant tumors. Even in modern times with advanced pharmaceutical sciences, the 5-year survival rate of lung cancer patients, especially solid tumor patients other than blood cancer patients, is less than 50%. About two-thirds of all cancer patients are diagnosed at an advanced stage, and most die within two years of cancer diagnosis. This poor result in cancer treatment is not only a problem of treatment methods, but more importantly, on the one hand, early diagnosis of cancer is very difficult, and it is not easy to achieve accurate diagnosis even in advanced ca...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12Q1/6869
CPCC12N15/1096C12Q1/6869C40B50/06C12Q2525/191C12Q2535/122
Inventor 赵鑫周鑫兰吴逵侯勇
Owner SHENZHEN HUADA GENE INST
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