Sequencing library and preparation and application thereof

A sequencing library and sequencing technology, applied in the field of sequencing library and its preparation and application, can solve the problem that the accuracy of DNA sequencing cannot meet the actual needs

Active Publication Date: 2017-04-05
AGRI GENOME INST OF SHENZHEN CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the problem that the accuracy of DNA sequencing in the prior art cannot meet the actual needs, the present invention provides a sequencing library and its preparation and application

Method used

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  • Sequencing library and preparation and application thereof
  • Sequencing library and preparation and application thereof
  • Sequencing library and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Example 1: Construct a whole-genome DNA library (Illumina platform) according to the above scheme 1 (double gap scheme)

[0095] 1) DNA fragmentation

[0096] Instruments and reagents used:

[0097] Ultrasonic interrupter: Covaris: S2 Focused-ultrasonicator

[0098] Interruption tube: Covaris Microtube 6x16mm, catalog#: 520045

[0099] QIAGEN MinElute Gel Extraction Kit(250), Catalog#:28606

[0100] Takara 20bp DNA Ladder (Dye Plus), Takara Code, 3420A

[0101] 5 μg of purified PhiX174 genomic DNA was broken into 150-200 bp with an ultrasonic breaker (Covaris S2Focused-ultrasonicator) (Intensity: 5, Duty Cycle: 10%, Cycles per Burst: 200, Temperature: 4°C, time: 60s ,number ofcycles:5), the interruption system is 50μl.

[0102] 4% agarose gel electrophoresis (80V, 70min; 1×TAE), gel cutting and recovery (QIAGEN MinEluteGel Extraction Kit) 60-90bp fragment (Takara 20bp DNA Ladder), the recovery steps are detailed in the QIAGEN MinElute Gel Extraction Kit manual.

...

Embodiment 2

[0185] Example 2: Construction of a genome-wide DNA library according to the above-mentioned scheme two (here, the three-gap scheme is taken as an example)

[0186] (1) DNA fragmentation, adding A to the end, connecting adapters, single-strand circularization steps are the same as in Example 1.

[0187] (2) Complementary strand synthesis

[0188] New England Biolabs: Klenow Fragment (3'→5'exo-), Catalog#:M0212S

[0189] New England Biolabs:USER TM Enzyme, Catalog #: M5505S

[0190] NEB buffer 4: 2μl

[0191] primer (UO-p1-2, 10uM): 1μl

[0192] DNA: 15.8 μl

[0193] 95°C for 3 minutes, and immediately placed on ice for 3 minutes.

[0194] When done add:

[0195] 2.5mM dNTPs: 0.5μl

[0196] 100X BSA: 0.2 μl

[0197] Klenow Fragment (3'→5'exo-): 1μl

[0198] Total 20μl

[0199] 20°C for 30 minutes, 75°C for 20 minutes.

[0200] USER TM Enzymes: 1μl

[0201] 37°C, 30min, 50°C, 5min, immediately put on ice.

[0202] The product was purified using Agencourt AMPure ...

Embodiment 3

[0205] Example 3: Construction of a whole genome DNA library according to the above-mentioned scheme three (double-stranded circularization scheme, the linker contains a notch to be cut)

[0206] (1) DNA fragmentation (~700bp, fragmentation conditions: duty cycle: 5%, intensity: 3, cyclesper burst: 200, time: 75s)), fill in the end and add A, connect the adapter with the same example 1, the adapter sequence It is: UO-A2, which is formed by annealing the following two sequences:

[0207] 5'-AGCACGTACGACTGAUCT-3' (SEQ ID NO: 5)

[0208] 5'-pGATCAGTCGTACGTGCT-3' (SEQ ID NO: 6)

[0209] (2) terminal phosphorylation

[0210] 44μl DNA, 10U T4PNK (T4Polynucleotide Kinase, NEB, M0201S), 50mMTris-HCl pH 7.5, 10mM MgCl2, 1mM ATP, 10mM DTT, 37°C for 30min, the product was purified with 1XAmpure XP magnetic beads.

[0211] (3) Double-strand cyclization

[0212] Quick Ligation Module(NEB,E6056S)

[0213] DNA: 35 μl

[0214] T4quick ligase: 5 μl

[0215] 5Xligase buffer: 10μl

[0...

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Abstract

The invention discloses a sequencing library. The library comprises a nucleotide sequence. The nucleotide sequence comprises a connecting sequence and two target sequences. Two ends of the connecting sequence are separately connected to the target sequences and the target sequences are direct repeat sequences. The invention further discloses preparation and an application of the sequencing library. The sequencing library solves the problem that the DNA sequencing accuracy in the prior art cannot satisfy the actual demand.

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. For example: in the process of microbial mutagenesis, how to monitor the mutation frequency distribution pattern caused by a certain mutagen under different mutagenic concentrations, so as to effectively optimize the mutagenesis system and improve the efficiency of mutagenesis; Screen a strain of target bacteria with a target mutation; detect whether there is a potential cancer-causing mutation site...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/10C12Q1/68C40B40/06C40B50/06
CPCC12N15/1093C12Q1/6806C12Q2525/191C40B40/06C40B50/06C12N15/10C12Q1/68C12N15/1065C12N15/1068C12N15/1086C12N15/1089C12N2310/3517C12N2320/10C12Q1/686C12Q1/6869C12Q2521/501C12Q2521/514C12Q2600/16C12Q2600/166
Inventor 阮珏王开乐
Owner AGRI GENOME INST OF SHENZHEN CHINESE ACADEMY OF AGRI SCI
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