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DNA index and application thereof to construction and sequencing of mate-paired indexed library

A technology of paired ends and tag sequences, applied in recombinant DNA technology, DNA/RNA fragments, libraries, etc., can solve the problems that cannot meet the increasing sequencing throughput

Active Publication Date: 2012-09-26
WUXI QINGLAN BIOLOGICAL SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the fourth version of the existing SOLiD sequencing chip can be divided into 8 regions at most, that is, each sequencing chip can sequence up to 8 paired-end library samples, which is far from meeting the increasing sequencing throughput. need

Method used

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  • DNA index and application thereof to construction and sequencing of mate-paired indexed library
  • DNA index and application thereof to construction and sequencing of mate-paired indexed library
  • DNA index and application thereof to construction and sequencing of mate-paired indexed library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Preparation of Label Cap Adapters

[0063] In this example, the Index1 LMP cap linker was synthesized by taking the Index1 sequence in Table 2 as an example.

[0064] a) Synthesize the two oligonucleotide sequences required to prepare the tag-cap adapter:

[0065] Index1 LMP cap adapter-a: 5'-ACAGCAGGGAAG-3' (SEQ ID NO: 31);

[0066] Index1 LMP cap linker-b: 5'-phos-CTTCCCTGCTGTAC-3' (SEQ ID NO: 32).

[0067] b) Dilute dry powder or film oligonucleotides to 125 μM.

[0068]c) Mix 125 μM Index1 cap adapter-a solution, 125 μM Index1 cap adapter-b solution, and 5X T4 ligase buffer (Invitrogen) in a volume ratio of 2:2:1, and dispense into PCR tubes, 100 μl each .

[0069] d) In thermal cycler (96-well Annealing hybridization was performed on the PCR System 9700) according to the following procedure.

[0070]

[0071] e) Take out the annealed double-stranded Index1 cap adapter and store it at -20°C for future use.

[0072] Similarly, prepare Index2-8LM...

Embodiment 2

[0073] Example 2: Construction of 2×50bp paired-end tag library

[0074] In this example, the genomic DNA of human blood mononuclear cells was used as an example to prepare a 2×50bp paired-end tag library, and the construction procedure refers to figure 1 .

[0075] 2.1 Main reagents

[0076] Unless otherwise noted, the relevant protein solutions, buffers, adapters or primer sequences in this example are all from the kit Applied Biosystems SOLiD TM Mate-PairedLibrary Oligo kit (4400468) or Applied Biosystems SOLiD TM Long Mate-Paired Library Construction kit(4443474).

[0077] 2.2 Experimental steps

[0078] Operation steps refer to Applied Biosystems SOLiD TM 4 System Library Preparation Guide P / N 4445673, section 3.1.

[0079] 1) Detection of DNA sample: no less than 20 μg, electrophoresis on 1% agarose gel for 40 minutes (130V) to detect DNA integrity; RNA and protein contamination are not allowed in the sample.

[0080] 2) Use the Hydroshear method to break the s...

Embodiment 3

[0101] Example 3: Hybrid Sequencing of Paired-End Indexed Libraries

[0102] 3.1 Main reagents

[0103] Unless otherwise noted, the reagents involved in this example are from Applied Biosystems.

[0104] 3.2 Experimental steps

[0105] 1) Mixed library

[0106] The Index1-4 library constructed according to the procedure shown in Example 2 was mixed according to the same amount of substances as library 9; the Index5-8 library constructed according to the procedure shown in Example 2 was mixed according to the same amount of substances as library 10; The Index1-8 library constructed according to the procedure shown in Example 2 was mixed to form library 11 in equal amounts.

[0107] 2) Amplification

[0108] Libraries 9-11 were used respectively, according to the emPCR standard procedure provided by Applied Biosystems (Applied Biosystems SOLiD TM 3 System Templated Bead Preparation Guide P / N4407421B) for emulsion PCR (emPCR) to obtain magnetic beads with template strands. ...

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Abstract

The invention provides a set of DNA index and application thereof to construction and sequencing of mate-paired indexed library, and the DNA index has a sequence selected from SEQ ID NO:1-24. The invention also provides a method for construction and sequencing of the mate-paired index library. The method employs two independent sequencing reactions to realize mixed sequencing on a plurality of mate-paired indexed libraries in a singe sequencing chip, so as to accelerate high flux sequencing, and reduce time, reagent cost and output cost of unit data.

Description

technical field [0001] The present invention relates to the second generation of high-throughput sequencing, especially the field of hybrid sequencing of paired-end libraries. More specifically, the present invention relates to DNA tags and their use in the construction and sequencing of paired-end tagged libraries. Background technique [0002] Paired-end library (mate-paired library) sequencing refers to the construction of a large fragment library to obtain the sequences at both ends of a large span (2-10kb) fragment. The sequences obtained from both ends of a large span play a very important role in the assembly of large or complex genomes and the discovery of genome structural variations, and are especially suitable for De novo sequencing projects. Currently, the paired-end library preparation method provided by the ABI SOLiD sequencing platform (Applied Biosystems SOLiD TM 4 System Library Preparation Guide P / N 4445673) such as figure 1 As shown, it includes steps:...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68C40B50/06C40B40/06
CPCC40B50/06C12Q1/6806C12Q1/68C40B40/06C12N15/11C12Q2521/501C12Q2525/191
Inventor 程磊
Owner WUXI QINGLAN BIOLOGICAL SCI & TECH
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