Improved sequencing library and preparation and application thereof

A technology for sequencing libraries and sequences to be tested, which is applied in the direction of DNA preparation, microbial measurement/inspection, biochemical equipment and methods, etc. It can solve the problems of DNA molecules that cannot be effectively balanced and amplified, and achieve the effect of improving balance

Active Publication Date: 2016-03-23
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

[0004] In order to solve the problem that circularized DNA molecules cannot be effectively balanced and amplified in the prior art, the embodiment of the present invention provides an improved sequencing library and its preparation and application

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] According to the scheme 1, the sequence to be tested and the tag sequence are constructed in the same direction as the concatemer library (IlluminaSolexa platform)

[0033] Circular DNA formation and replication part:

[0034] DNA fragmentation

[0035] Instruments and reagents used:

[0036] Ultrasonic interrupter: Covaris: S2Focused-ultrasonicator

[0037] Interruption tube: CovarisMicrotube6x16mm, catalog#: 520045

[0038] Agarose: Promega, Agarose, LE, Analytical Grade, catalog #: V3121

[0039] Electrophoresis instrument power supply: Beijing Liuyi Instrument Factory, DYY-7C type

[0040] Electrophoresis tank: Beijing Liuyi Instrument Factory, DYCP-31DN electrophoresis tank

[0041] QIAGENMinEluteGelExtractionKit(250), Catalog#:28606

[0042] Takara20bpDNALadder (DyePlus), TakaraCode, 3420A

[0043] Use an ultrasonic interrupter (CovarisS2Focused-ultrasonicator) to break 1ug of purified genomic DNA into 300bp (Intensity: 4, DutyCycle: 10%, CyclesperBurst: 20...

Embodiment 2

[0258] According to protocol 1, construct the peripheral blood cell-free DNA test sequence and tag sequence direct alternating concatemer library (Illumina sequencing platform)

[0259] Cell-free DNA was extracted from peripheral blood and its fragment size was detected.

[0260] Instruments and reagents used:

[0261] QIAGEN: QIA & Circulating Nucleic Acid Kit, catalog #: 55114

[0262] Agilent: 2100 bioanalyzer

[0263] Take 2ml of plasma, use QIAampCirculatingNucleicAcidKit of QIAGEN to extract the DNA (cell-freecirculatingDNA) in the plasma, and elute with 20ulddH2O (see the kit manual for the extraction method). Agilent's 2100bioanalyzer was used to detect the size distribution of the extracted fragments. From the results, the size of the 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 patients is concentrated around 164b...

Embodiment 3

[0273] Construct the test sequence and the tag sequence direct alternating concatemer library according to the second scheme (Illumina sequencing platform)

[0274] step:

[0275] DNA Fragmentation: See Example 1.

[0276] End Filling: See Example 1.

[0277] Add A to the end and connect the tag sequence: see Example 1.

[0278] DNA single-strand circularization: see Example 1.

[0279] rolling circle amplification

[0280] Instruments and reagents used:

[0281] PCR instrument: Eppendorf: Mastercyclerpros

[0282] New England Biolabs: phi29 DNA Polymerase, Catalog #: M0269L

[0283] Single-stranded circularized DNA: 5.7ul

[0284] phi29DNAPolymeraseReactionBuffer: 2ul

[0285] Primer UO-a3 (10pmol): 1ul

[0286] wxya 2 O: 8.9ul

[0287] A total of 17.6ul, 95 ℃ 3min, immediately placed on ice for 3min. When done add:

[0288] 10mMdNTP: 1ul

[0289] 100XBSA: 0.4ul

[0290] phi29DNA Polymerase (10U / ul): 1ul

[0291] Total: 20ul

[0292] 8h at 30°C, 10min at 65°C...

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Abstract

The invention provides a sequencing library. An insertion fragment in the sequencing library is a conjugate alternate series body of a sequence to be measured and a label sequence. Preparation of the conjugate alternate series body of the sequence to be measured and the label sequence comprises the steps that the sequence to be measured is connected with the label sequence, and single-chain circulation and rolling circle replication are carried out, wherein rolling circle replication is completed in a reaction solution wrapped by oil drops. The invention further provides a preparing method of the sequencing library and a sequencing method. According to the sequencing library and the sequencing method, DNA amplification errors and sequencing errors can be effectively eliminated under any sequencing depth, and thus mutation existing on DNA molecules is detected super-precisely. The sequencing library is suitable for construction of microscale DNA short fragments and even a single-chain DNA sequencing library.

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 sequences. Although 1% error rate is tolerable in some applications, in many cases, this 1% error has covered up a lot of real information, and has become an obstacle to many researches. It is very necessary to construct a method that can quickly, efficiently and accurately construct a DNA sequencing library. [0003] A sequencing library invented by the inventor, as shown in the patent application document No. 201310651462.5, solves the above-mentioned problems existing in the prior art. However, due to the great sequence preference of rolling circle amplification, some circular am...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/10
Inventor 王开乐阮珏吕雪梅吴仲义
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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