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Preparation of MGI platform transposase double-terminal tag library

A transposase and library technology, which is applied in the field of transposase library preparation, can solve the problem of mixed testing of MGI transposase library and MGI non-transposase library, and achieve the effect of saving time and cost, and the process is simple and fast

Active Publication Date: 2022-08-02
VAZYME BIOTECH NANJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to provide a method for preparing and sequencing a transposase double-end tag (barcode) library on the MGI platform, which solves the problem that the existing MGI transposase library cannot be mixed with the MGI non-transposase library, and is more convenient and efficient
Contents of the invention

Method used

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  • Preparation of MGI platform transposase double-terminal tag library
  • Preparation of MGI platform transposase double-terminal tag library
  • Preparation of MGI platform transposase double-terminal tag library

Examples

Experimental program
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reference example

[0069] Conventional / standard, i.e. non-transposed, dual-indexed rolling circle sequencing libraries with features such as Figure 7 Library structure shown. See CN111910258A.

[0070] In one embodiment, the full sequence of the double-ended library splint is

[0071] 5'-CTGATAAGGTCGCCATGCCTCTCAGTACGTCAGCAGTT-3' (SEQ ID NO: 6), double-end library splint sequence 1 (ie, the 3' part of the double-end library splint full sequence) is 5'-CTCTCAGTACGTCAGCAGTT-3' (SEQ ID NO: 7), Double-ended library splint sequence 2 (ie, the 5' portion of the complete double-ended library splint sequence) is 5'-GCATGGCGACCTTATCAG-3' (SEQ ID NO: 8).

[0072] The full sequence of the single-ended library splint is

[0073] 5'-CAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT-3' (SEQ ID NO: 9). The corresponding looping primer sequence is 5'-GCCATGTCGTTCTGTGAGCCAAGG-3' (SEQ ID NO: 10).

[0074] Tag sequence 1 and tag sequence 2 are each 10 consecutive nucleotides.

[0075] The insert primer binding sequ...

Embodiment 1

[0077] In this example, the improved second-generation sequencing of human blood gDNA and E. coli gDNA samples is performed, that is, Tn5 transposase performs NGS second-generation library construction and sequencing on the samples, and the off-machine data is split, filtered, and compared. Assess the sequencing quality of this case. The library-building reagents used in this example are from the TruePrep Flexible DNA Library Prep Kit for MGI (#TDM504) from Nanjing Novizan.

[0078] 1. Fragmentation

[0079] (1) Thaw 5×TTBL at room temperature, invert and mix well before use. Make sure that the 6×TSB and TWB are at room temperature, and flick the tube wall to confirm that there is no precipitation; if there is any precipitation, heat at 37°C and vortex to mix until the precipitation dissolves.

[0080] (2) Prepare the following reaction system in a PCR tube

[0081] component volume 5×TTBL 10μl DNA to be tested (1ng) 1μl TNB 10μl ddH 2 ...

Embodiment 2

[0169] Same as Example 1, the only difference is:

[0170] Barcode 1' corresponding to primer Barcode 1 in Example 1 is the sequence shown in SEQ ID NO: 14, and Barcode 2' corresponding to primer Barcode 2 in Example 1 is the sequence shown in SEQ ID NO: 15.

[0171] SEQ ID NO: 15

[0172] 5’-GCATGGCGACCTTATCAGNNNNNNNNNNTCGTCGGCAGCGTCAGAT-3’

[0173] SEQ ID NO: 14

[0174] 5’-CTCTCAGTACGTCAGCAGTTNNNNNNNNNNCAACTCCTTGGCTCACAGAACGACATGGCGTCTCGTGGGCTCGGAGA-3’

[0175] Sequencing primer sequences include the following sequences:

[0176]

[0177]

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Abstract

The invention provides a method for preparing a transposition type double-label rolling circle sequencing library compatible with a non-transposition type double-label rolling circle sequencing library, and a reagent used in the method, in particular to an oligonucleotide pair.

Description

technical field [0001] The invention relates to the technical field of gene sequencing, in particular to a method for preparing a transposase library and a second-generation sequencing method. Background technique [0002] High-throughput sequencing technology, also known as Next-generation "sequencing technology", which can be abbreviated as NGS, refers to the parallel sequencing of hundreds of thousands to millions of DNA molecules at a time. The technology of sequence determination has high accuracy and large throughput. [0003] The conventional second-generation sequencing library construction method is to fragment the double-stranded DNA by physical or chemical methods, end repair, connect adapters, and purify the library after PCR amplification. Tn5 transposase is a kind of transposase, which can transform the tedious steps of DNA fragmentation, end repair and adapter ligation into a simple enzymatic reaction, and is widely used in the construction of next-generation...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/6806C40B50/06C12Q1/6869
CPCC12Q1/6806C40B50/06C12Q1/6869C12Q2525/191C12Q2521/50C12Q2523/308C12Q2531/125C12Q2535/122
Inventor 聂俊伟瞿志鹏曹林张宇朱涵雪
Owner VAZYME BIOTECH NANJING