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Bubble linker and its application in nucleic acid library construction and sequencing

A single-stranded circular library and bubble-shaped technology, applied in nucleotide libraries, protein nucleotide libraries, libraries, etc., can solve the problems of many by-products, low connection efficiency, and no relevant literature reports

Active Publication Date: 2020-05-05
MGI TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the currently reported adapter ligation methods for nucleic acid sequencing libraries often have low connection efficiency and many by-products.
In addition, since the CG sequencing platform uses single-stranded circular libraries for sequencing, the double-stranded linear libraries usually constructed by mainstream sequencing platforms are not suitable for CG sequencers
For the construction method of single-stranded circular library in nucleic acid sequencing, there is no relevant literature report

Method used

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  • Bubble linker and its application in nucleic acid library construction and sequencing
  • Bubble linker and its application in nucleic acid library construction and sequencing
  • Bubble linker and its application in nucleic acid library construction and sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Construction of RNA Libraries Using Bubbly Nucleotide Linkers

[0096] Specific experimental steps (see figure 1 Process steps shown in ):

[0097] Specific experimental steps:

[0098] 1. Purify mRNA

[0099] 1) Take standard universal human reference RNA (Agilent) (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.

[0100] 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, and put the total RNA prepared in the first step Add to the tube and let stand at room temperature for 5min.

[0101] 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 washingbuffer. Take a new non-stick EP tube and add 50...

Embodiment 2

[0169] Example 2 compares the PCR efficiency of using bubble joints and other types of joints in library construction

[0170] Specific experimental steps:

[0171] Adopt the same procedure as in Example 1, wherein one kind of joint uses the foam joint, and the control joint uses the mating joint. Complete the PCR amplification and purification in step 6, and then detect the amount of the purified PCR product.

[0172] The PCR template concentration and recovery concentration were measured using Qubit dsDNA Assay Kit.

[0173] The experimental results are shown in Table 1

[0174] Connector name Mating connector bubble joint Amount of PCR template (ng) 10 10 Recovered product concentration (ng / ul) 5.66 53 Total recovered product (ng) 226.4 2120 PCR efficiency 1.366 1.709

[0175] Remarks: PCR efficiency = (total PCR output / template input amount) × (1 / cycle number)

[0176] From the above results, it can be seen that the PCR eff...

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PUM

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Abstract

Provided are a bubble adapter and a single-stranded circular library constructed using the adapter, the library can be used for RNA sequencing and other sequencing platforms relying on single-stranded circular libraries, and has the advantages of high sequencing throughput, high accuracy and easy operation The advantages.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a foam joint and a method for constructing a nucleic acid library and a sequencing method using the joint. 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 second-generation sequencing is represented by Roche / 454 pyrosequencing, Illumina / Solexa polymerase synthesis sequencing and ABI / SOLiD ligase sequencing. Their common feature is high sequencing throughput. 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 per hour can reach 50Gb, which is 10-10% higher than that of the current mainstream sequencing platforms. 25 times. In terms of haploid read length, amon...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C40B50/06C40B40/06C12Q1/6869
CPCC12Q1/6806C12N15/66C12Q2525/191B01J2219/00529C40B60/14C12N15/1089C12Q2525/186C12N15/1093C12Q2521/101C12Q2521/525C12Q2535/122C40B40/08C40B50/06B01J19/0046C12N15/10C12Q1/6811C12Q1/6837C12Q1/6853C12Q1/686C12Q2565/537C12Q1/6855C12Q1/6874C12N9/22C12N9/93C12N11/06C12N15/1065C12N15/1082C40B50/14
Inventor 江媛郭晶纪晓钧耿春雨田凯赵霞徐怀前章文蔚蒋慧拉多杰·德马纳克
Owner MGI TECH CO LTD
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