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Transposase complex containing unique molecular identifier sequence and application of transposase complex

A technology of molecular tags and tag sequences, which is applied in the field of sequencing, can solve the problems of commercial sequencing restrictions, occupying index sequences, etc., and achieve the effects of facilitating commercial promotion and application, improving efficiency, and improving utilization

Pending Publication Date: 2021-01-22
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Patents CN103938277B and CN110628889A disclose two methods for constructing sequencing libraries containing random bases (i.e. UMI), but the way of reading random bases is through index primers, which occupy the index of different samples. sequence, which brings limitations to commercial sequencing

Method used

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  • Transposase complex containing unique molecular identifier sequence and application of transposase complex
  • Transposase complex containing unique molecular identifier sequence and application of transposase complex
  • Transposase complex containing unique molecular identifier sequence and application of transposase complex

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Construction (assembly) of transposase complexes with unique molecular tag sequences:

[0073] 1. Designing Oligonucleotide Linkers

[0074] ME-A6 cgacgctcttccgatctnnnnnnagatgtgtataagagacag MErev [phos]ctgtctcttatacacatct

[0075] Note: ME-A6 is the first strand of the oligonucleotide adapter, where CGACGCTCTTCCGATCT is the universal adapter sequence, NNNNNN is the UMI sequence, and AGATGTGTATAAGAGACAG is the ME sequence, which is reverse complementary to the second strand MErev required to form a complete adapter.

[0076] 2. Transposase Complex Assembly

[0077] a. Mix MErev and ME-A6 according to 1:1, mix 20μl at a time;

[0078] b. After the mixed primers were denatured at high temperature (95°C for 3 minutes), the temperature was gradually lowered and annealed to 12°C to obtain the adapter MEDS-A6 at the 5' sticky end, which is the adapter used for assembly.

[0079] c. Mix the linker synthesized above with Tn5 enzyme (Tn5 enzyme can be pur...

Embodiment 2

[0085] The kit includes the complex composition obtained in Example 2, buffer, PCR primers, PCR enzyme and PCR amplification buffer;

[0086] Transposition reaction buffer system is 50mM Tris-HCl, 25mM MgCl 2 and 50% v / v DMF or other suitable buffer for Tn5 transposition reaction.

[0087] PCR primers are:

[0088]

[0089] Wherein the underlined part is an alternative barcode sequence.

[0090] PCR enzymes are high-fidelity enzymes such as High-Fidelity 2X PCR Master Mix.

[0091] The complex composition preparation method in the above kit is as follows:

[0092] 1. Designing Oligonucleotide Adapter Sets

[0093] The linker sequence required for the assembly of Tn5 fused with UMI was synthesized, as shown in Table 2.

[0094] Table 2. Tn5 linker sequence primers

[0095]

[0096]

[0097] Note: The bases in lowercase letters in ME-A25 are the barcode sequences that adjust the base balance. ME-B is the sequence described in the literature (Jason DBuenrostro e...

Embodiment 3

[0104] Using rice leaves as materials, the kit of Example 2 was used to construct an ATAC-seq library containing a UMI linker (hereinafter referred to as UMI-ATAC-seq):

[0105] Step S1, material preparation and library construction ( Figure 4 b)

[0106] a. Preparation of cell nuclei from rice leaves: Take fresh rice leaves, add 500 μL of cell lysate, chop quickly with a blade, filter and add 0.1% DAPI for staining. 100,000 cell nuclei were sorted by flow cytometry into a 1.5mL centrifuge tube, and the cell nuclei were collected by centrifugation, and kept on ice for later use.

[0107] b. Transposition reaction and purification: configure the reaction system on ice according to the following ratio: 2 μL complex composition, 8 μL 5x tagmentation buffer, 30 μL ddH 2 O. After mixing by pipetting, incubate at 37°C for 30min. After the reaction was completed, it was purified with TaKaRa MiniBEST DNA Fragment Purification Kit (No.9761).

[0108] c.PCR amplification: using ...

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Abstract

The invention discloses a transposase complex containing a unique molecular identifier sequence and application of the transposase complex. The transposase complex is a complex formed by assembling oligonucleotide linkers and transposase. A next-generation sequencing library constructed by utilizing the transposase complex containing the UMI can effectively distinguish transposase insertion repeatevents and repeat reads caused by PCR, so that the utilization rate of sequencing data, the reliability of sequence variation and frequency identification of the sequence variation, the accuracy of gene expression and chromatin accessibility quantification, and the identification sensitivity of the footprint of the DNA binding protein are improved.

Description

technical field [0001] The invention relates to the technical field of sequencing, in particular to a transposase complex containing a unique molecular tag sequence (UMI) and an application thereof. Background technique [0002] Next Generation Sequencing (NGS) technology has profoundly affected biological research and clinical diagnosis. The construction of sequencing library is the basis of NGS. Since 2010, transposase Tn5 has been widely used to construct DNA sequencing libraries based on Illumina's sequencing platform. The principle is: Tn5 is a bacterial transposon, which was first discovered in Escherichia coli. After modification, Tn5 transposase can combine with ME (mosaic end) double-stranded sequence with a length of 19 bp, and insert it into large double-stranded DNA fragments almost randomly. Therefore, as long as the adapter is synthesized together with the ME sequence, then incubated with Tn5 transposase to form a transposition complex, and then incubated wi...

Claims

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Application Information

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IPC IPC(8): C12N9/12C07K19/00C12Q1/6806C12N15/10C40B50/06
CPCC12N9/1241C07K19/00C12Q1/6806C12N15/1093C40B50/06C12Q2531/113C12Q2525/191C12Q2563/179Y02A50/30
Inventor 谢为博
Owner HUAZHONG AGRI UNIV
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