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Oligonucleotide adapters and their application in the construction of single-strand circular libraries for nucleic acid sequencing

A technology of oligonucleotide and sequencing library, which is applied in the field of oligonucleotide linker and the use of the linker to construct nucleic acid sequencing single-stranded circular library, which can solve the problem of increasing operational complexity, affecting experimental efficiency, and not having satisfactory single-stranded The construction method of circular library and other issues

Active Publication Date: 2019-12-31
MGI TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The RNA-seq sequencing technology provided by Illumina, represented by the Solexa sequencing platform, works well, but because it constructs a double-stranded linear sequencing library, this type of library is not suitable for the CG sequencing platform, and there are also some problems that affect the efficiency of the experiment. Complicated matters, such as the need to use ethanol precipitation to recover the sample after the interruption, in order to carry out the subsequent reverse transcription reaction, this step not only affects the speed of the experiment, but also increases the complexity of the operation
[0004] At present, there is no satisfactory method for constructing a single-stranded circular library for RNA sequencing in this field. Therefore, there is an urgent need in the field to develop a method for efficiently preparing high-quality RNA sequencing that can be applied to a CG sequencing platform. Single-stranded circular library method

Method used

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  • Oligonucleotide adapters and their application in the construction of single-strand circular libraries for nucleic acid sequencing
  • Oligonucleotide adapters and their application in the construction of single-strand circular libraries for nucleic acid sequencing
  • Oligonucleotide adapters and their application in the construction of single-strand circular libraries for nucleic acid sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0135] Example 1 Construction of RNA single-stranded circular library

[0136] Specific experimental steps (see figure 1 Process steps shown in ):

[0137] 1. mRNA extraction and purification

[0138] 1) Take standard universal human reference RNA 3ug into an RNase-free tube and dilute to 50μl with DEPC water. Mix well, denature at 65°C for 5 minutes to open up secondary structures, and immediately place samples on ice.

[0139] 2) Pipette 15μl Dynalbeads Oligo(dT) 25 Put the magnetic beads in a 1.5ml non-stick EP tube, wash the magnetic beads twice with 100μl binding buffer, resuspend the magnetic beads in 50μl binding buffer, add the total RNA prepared in the first step to the tube in room temperature for 5 minutes.

[0140] 3) Place the non-stick-EP tube on the MPC (magnetic separator) for 2 minutes, remove the supernatant, and wash the magnetic beads twice with 200 μl of washing buffer. Take a new non-stick EP tube and add 50 μl of binding buffer.

[0141] 4) Add 50...

Embodiment 2

[0222] The library preparation steps 1-10 in Example 1 were repeated, with the difference that the standard universal human reference RNA in Example 1 was replaced by conventional human lymphocytes (YH cells) as the source of total RNA. The total RNA of Yanhuang cells was extracted with QIAGEN RNA extraction kit. Before the extraction and purification of mRNA, the total RNA was pretreated. The pretreatment steps are as follows:

[0223] 1. DNase digestion of total RNA

[0224] Take the total RNA sample, put it in an RNase-free EP tube, and carry out DNA digestion according to the following system.

[0225]

[0226] Digest at 37°C for 10 minutes.

[0227] Add 2μl 1mM EDTA solution, denature at 75°C for 10min, and cool on ice.

[0228] 2. Purification of digested RNA

[0229] Add the product (22 μl) of the above steps to 1.8 times the volume (40 μl) of RNA clean magnetic beads (need to equilibrate at room temperature for 30 minutes in advance). Place at room temperature f...

Embodiment 3

[0242] The DNA of Hela tumor cells was extracted with QIAGEN High Quality DNA Extraction Kit. Then repeat steps 4 to 9 in Example 1, 6% TBE denatured gel detection.

[0243] see results Figure 7 , the results show that it is feasible to use the method of the present invention to prepare a DNA sequencing library.

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Abstract

The present invention provides an oligonucleotide linker, and particularly relates to uses of the oligonucleotide linker in construction of a single-chain cyclic library in nucleic acid sequencing, and further in sequencing platforms requiring single-chain cyclic libraries. The oligonucleotide linker of the present invention has advantages of high sequencing throughput, high accuracy, and easy operation.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to an oligonucleotide linker, a method for constructing a single-strand circular library for nucleic acid sequencing by using the linker, and its application. Background technique [0002] Second-generation sequencing technology, also known as next-generation sequencing technology, is named after the first-generation sequencing technology represented by Sanger sequencing. The three mainstream sequencing technologies in second-generation sequencing are Roche / 454 pyrosequencing, Illumina / Solexa polymerase sequencing by synthesis, and ABI / SOLiD ligase sequencing technologies. Compared with these mainstream sequencing platforms, the Complete Genomics (CG) sequencing platform has the highest throughput, which can generate 9.9TB of data per run, and the output can reach 50Gb per hour, which is 10-25 times that of the current mainstream sequencing platforms . CG sequen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C40B50/06C40B40/06
Inventor 郭晶纪晓钧祝珍珍
Owner MGI TECH CO LTD
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