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Sequencing library, preparation and application thereof

一种测序文库、文库的技术，应用在测序文库及其制备和应用领域，能够解决DNA测序准确率不能满足实际需求等问题，达到扩增效率高的效果

Active Publication Date: 2015-06-10
BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In order to solve the problem that the accuracy of DNA sequencing cannot meet the actual needs in the prior art, the embodiment of the present invention provides a sequencing library and its preparation and application

Method used

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  • Sequencing library, preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Construct the test sequence and tag sequence direct alternating concatemer library (Illumina platform) according to scheme 1

[0037] DNA fragmentation

[0038] Instruments and reagents used:

[0039] Ultrasonic interrupter: Covaris: S2 Focused-ultrasonicator

[0040] Interruption tube: Covaris Microtube 6x16mm, catalog #: 520045

[0041] Agarose: Promega, Agarose, LE, Analytical Grade, catalog #: V3121

[0042] Electrophoresis instrument power supply: Beijing Liuyi Instrument Factory, DYY-7C type

[0043] Electrophoresis tank: Beijing Liuyi Instrument Factory, DYCP-31DN electrophoresis tank

[0044] QIAGEN MinElute Gel Extraction Kit (250), Catalog #: 28606

[0045] Takara 20 bp DNA Ladder (Dye Plus), Takara Code, 3420A

[0046] 1ug purified genomic DNA was fragmented into 300bp with an ultrasonic fragmentation instrument (Covaris S2 Focused-ultrasonicator) (Intensity: 4, Duty Cycle: 10%, Cycles per Burst: 200, Temperature: 4°C, time: 60s, number of cycles: 2)...

Embodiment 2

[0342] According to the protocol 1, the peripheral blood cell-free DNA sequence to be tested and the tag sequence are constructed in the same direction as the concatemer library (Illumina sequencing platform)

[0343] Cell-free DNA was extracted from peripheral blood and its fragment size was detected.

[0344] Instruments and reagents used:

[0345] QIAGEN: QIAamp Circulating Nucleic Acid Kit, catalog #: 55114

[0346] Agilent: 2100 bioanalyzer

[0347] Take 2ml of plasma, use QIAamp Circulating Nucleic Acid Kit of QIAGEN to extract the DNA (cell-free circulating DNA) in the plasma, and elute with 20ul ddH2O (see the kit manual for the extraction method). Agilent's 2100 bioanalyzer was used to detect the size distribution of the extracted fragments. From the results, the size of free DNA fragments in normal people is concentrated around 172bp, the distribution range is about (130bp-230bp), and the concentration is 0.354ng / ul. The size of free DNA fragments in liver cancer...

Embodiment 3

[0463] Construct the sequence to be tested and the tag sequence in the same direction as the alternate concatemer library (Illumina sequencing platform) according to the second scheme

[0464] step:

[0465] DNA fragmentation

[0466] Instruments and reagents used:

[0467] Ultrasonic interrupter: Covaris: S2 Focused-ultrasonicator

[0468] Interruption tube: Covaris Microtube 6x16mm, catalog #: 520045

[0469] Agarose: Promega, Agarose, LE, Analytical Grade, catalog #: V3121

[0470] Electrophoresis instrument power supply: Beijing Liuyi Instrument Factory, DYY-7C type

[0471] Electrophoresis tank: Beijing Liuyi Instrument Factory, DYCP-31DN electrophoresis tank

[0472] QIAGEN MinElute Gel Extraction Kit (250), Catalog #: 28606

[0473] Takara 20 bp DNA Ladder (Dye Plus), Takara Code, 3420A

[0474] 1ug purified genomic DNA was fragmented into 300bp with an ultrasonic fragmentation instrument (Covaris S2 Focused-ultrasonicator) (Intensity: 4, Duty Cycle: 10%, Cycles ...

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Abstract

Provided in the present invention is a sequencing library and a preparation method thereof, wherein the inserted fragment of the sequencing library is a concatemer in which the sequences to be tested and tag sequences are alternated along the same direction. Also provided is a sequencing method. The sequencing library and sequencing method, under any sequencing depth, can effectively remove errors in DNA amplification and sequencing so as to detect the mutations in DNA molecules accurately, and are suitable for the sequencing library construction of trace short DNA fragments or even single-strand DNA.

Description

technical field [0001] The invention relates to a sequencing library and its preparation and application. Background technique [0002] The development of second-generation sequencing technology has promoted the revolutionary development of biological and biomedical research. However, due to the characteristics of high-throughput sequencing itself, there are about 1% base errors in the measured sequence. Although a 1% error rate is tolerable in some applications, in many cases, this 1% error conceals a lot of real information and becomes an obstacle to many researches. For example: detecting whether there is a potential cancer-causing mutation site in a certain tissue or organ of a normal individual, detecting the heterogeneity of DNA composition in cancer cell populations and hidden small clone populations, using DNA mutations in each cell as a marker to trace The origin and division mode of the cell, accurately obtain the genotype in a highly heterozygous cancer populati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B40/06C40B50/06C12Q1/68
CPCC12Q1/6869C12Q2525/151C12N15/10C12N2310/51C12Q1/6844C12Q2537/155
Inventor 阮珏王开乐吕雪梅吴仲义
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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