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Asymmetric high-throughput sequencing linkers capable of effectively improving library construction efficiency, and application of linkers

A sequencing adapter, high-throughput technology, applied in the field of high-throughput sequencing

Inactive Publication Date: 2015-12-16
南京普东兴生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the above problems, the present invention provides an asymmetric high-throughput sequencing adapter and its application that can effectively improve the efficiency of library construction, so as to solve the problem of connecting the same adapter at both ends of the target DNA fragment during the library construction process.

Method used

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  • Asymmetric high-throughput sequencing linkers capable of effectively improving library construction efficiency, and application of linkers
  • Asymmetric high-throughput sequencing linkers capable of effectively improving library construction efficiency, and application of linkers
  • Asymmetric high-throughput sequencing linkers capable of effectively improving library construction efficiency, and application of linkers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] 1. Example 1: Construction of Tongue Cancer Genome 150bp Library

[0053] 1. Genomic DNA fragmentation (using CovarisS220 ultrasonic breaker)

[0054] (1) Take 2 μg of tongue cancer genomic DNA, and perform ultrasound interruption according to the set parameters, PeakincidentPower: 175, Dutyfactor: 10%, CyclesperBurst: 200, TreatmentTime(s): 430, Temperature(°C): 7, Waterlevel: 12, Sample Volume (μL): 130.

[0055] 2. 2% agarose gel electrophoresis (fragment selection)

[0056] (1) According to the DL500 molecular beacon, select fragments with a fragment size of 140-160bp for gel cutting and recovery. The agarose gel recovery kit used here is Tiangen agarose gel recovery kit;

[0057] 3. DNA fragments are subjected to end repair (the end repair kit used here is EpicentreEnd-it TM DNAEnd-RepairKit)

[0058] (1) Take 50ng of fragmented DNA of the above size for end repair: Fragmented DNA: 34 μL, 10×End-it buffer: 5 μL, End-itATP (10mM): 5 μL, End-itdNTPs (2.5mM): 5 μ...

Embodiment 2

[0081] 2. Example 2: Construction of Human Blood Circulating Nucleic Acid Library

[0082] 1. DNA fragment end repair

[0083] (1) Take 100 ng of circulating nucleic acid for end repair (the end repair kit used here is EpicentreEnd-itTMDNAEnd-RepairKit): Fragmented DNA: 34 μL, 10×End-it buffer: 5 μL, End-itATP (10 mM): 5 μL, End-itdNTPs (2.5mM): 5μL, End-itenzymemix: 1μL, Nuclease-freeWater: 0μL;

[0084] (2) Incubate at room temperature for 30 minutes;

[0085] (3) Purify the obtained product with magnetic beads, and the magnetic beads used here are BeckmanAgencourtAMPureXP nucleic acid purification magnetic beads.

[0086] 2. Add A to the 3' end of the DNA fragment that has been repaired

[0087] (1) Add A to the product (32 μL) obtained in the previous step: DNA: 32 μL, NEBBuffer: 25 μL, dATP (10 mM): 1 μL, Nuclease-freeWater: 9 μL, Klenowexo - : 3 μL;

[0088] (2) Incubate at 37°C for 30 minutes;

[0089] (3) Purify the obtained product with magnetic beads, and the m...

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Abstract

The invention relates to the field of high-throughput sequencing, and provides asymmetric high-throughput sequencing linkers for construction of an SOLiD sequencing system library, and a library construction method which is suitable for an SOLiD sequencing system and adopts the asymmetric high-throughput sequencing linker. The asymmetric high-throughput sequencing linkers are prepared by separately selecting one DNA single stranded from a P1 linker sequence and a P2 linker sequence which are inherent in the SOLiD sequencing system, and modifying and annealing the selected linkers. Asymmetric areas of the modified linkers contain binding sites of library amplification primers. The asymmetric high-throughput sequencing linkers provided by the invention are applied to the construction of the SOLiD sequencing system library, a phenomenon that two ends of one sequencing fragment are connected to the same linker during library construction in the prior art is avoided, and the library construction efficiency is effectively improved.

Description

technical field [0001] The present invention relates to the field of high-throughput sequencing, and more specifically, relates to an asymmetric high-throughput sequencing adapter applied to a SOLiD sequencing system, which can effectively improve the efficiency of library construction and its application. Background technique [0002] High-throughput sequencing technology has been widely used in various fields of biological research, and many biological problems have been solved with the help of high-throughput sequencing technology. The SOLiD sequencing system can sequence the sequencing library made by any method. Its biggest feature is the use of double-base coding technology, which has the function of error correction because it uses two bases to correspond to a fluorescent signal. Instead of the traditional one base corresponding to one fluorescent signal, each site will be detected twice, so the error rate is significantly reduced. After the sequencing library is con...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/10C12Q1/68C40B50/06
Inventor 沈双烨戴琳超陆祖宏
Owner 南京普东兴生物科技有限公司
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