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Double-label joint sequence for detection of tumor mutation and detection method

A linker sequence and double-label technology, which is applied in biochemical equipment and methods, microbial determination/testing, chemical library, etc., can solve the problems of inaccurate detection and inability to distinguish signal-to-noise ratio

Active Publication Date: 2016-11-09
AMOY DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] For mutation sites such as tumor cell mutations (somatic mutation) and other mutation sites, there is great heterogeneity between cells (mutation sites in each cell may be different), and the proportion of such mutations in the sample is very large. Low (less than 1%), this type of mutation cannot be distinguished using traditional bioinformatics methods (the signal-to-noise ratio between the systematic base error rate as noise and the signal of the tumor mutation site is too low), so the tumor site mutation is used Conventional sequencing methods cannot accurately detect

Method used

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  • Double-label joint sequence for detection of tumor mutation and detection method
  • Double-label joint sequence for detection of tumor mutation and detection method
  • Double-label joint sequence for detection of tumor mutation and detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0081] Embodiment 1: the preparation of Duplex linker

[0082] Connector primer P5, two primers of connector primer P7 (connector primer P5 is shown in the sequence obtained by connecting SEQ ID NO: 1 through the I5 index sequence of SEQ ID NO: 1; connector primer P7 is SEQ ID NO: 3 connected through the I7 index sequence SEQ ID NO: 4 is shown in the obtained sequence; wherein FFFFFFEEEEEDDDDDNNNNNNNNNNNNN is sequentially connected to the 5' end of SEQ ID NO: 3; synthetic manufacturer: Sangon Bioengineering (Shanghai) Co., Ltd.) with ddH 2 O (or TE buffer) diluted to 100uM;

[0083] Wherein FFFFF is the protection base of the enzyme cutting site, EEEEE is the enzyme cutting site, DDDDD is the positioning tag sequence, NNNNNNNNNNNN is the random molecular tag sequence, the I5 index sequence is selected from SEQ ID NO:5-12; the I7 index The sequence is selected from SEQ ID NO: 12-23.

[0084] At the same time, FFFFF / DDDDD / EEEEE / includes but is not limited to 5 identical bases;...

Embodiment 2

[0097] Embodiment 2: detection of plasma DNA mutation rate

[0098] In this embodiment: the protective base is TCTTCT; the enzyme cutting site sequence is (The positioning base is in the box, and the restriction site and the positioning base partially overlap); the molecular label is BDHVBDHV.

[0099] I5 and I7 can be: I501-I701, I502-I702, I503-I703, I504-I704, I505-I705, I506-I706, I507-I707, I508-I708, I501-I707, I502-I708, I503-I709 , I504-I710. (The base sequence corresponding to the serial number is shown in Table 1)

[0100] Sample selection and quality control: Take 5 plasma samples of lung cancer patients, use QIAGEN plasma DNA extraction kit to extract plasma DNA, use spectrophotometer to measure DNA sample purity (requires A260 / 280 between 1.8-20); then use Qubit2 .0 Measure DNA concentration (the total amount is between 5-15ng), use D1000chip (Agilent) to detect DNA sample fragment distribution (about 160-200bp) use digital PCR (Bio-rad) to measure tumor sampl...

Embodiment 3

[0122] Example 3: Cell line mutation rate detection

[0123] Two cell line DNAs, NCI-H1650 and HCT, were selected as experimental materials. NCI-1650 cell DNA was incorporated into HCT cell DNA at a mass ratio of 10%, 1%, and 0.1%, respectively. In addition, NCI-1650 and HCT cell 100% %DNA was taken as two samples respectively, correspondingly recorded as 10%, 1%, 0.1%, NCI-1650 and HCT groups. (NCI-1650 and HCT groups are only to determine the genetic background of the cell line DNA used for mixing ratios—that is, allelic locus information, such as heterozygous homozygosity, etc., through the sequencing information of these two samples, find out some Homozygous base sites, and then pick out the sites with different bases at the same site as the statistical analysis sites for other sample groups).

[0124] After the DNA samples were fully mixed, 2ug were taken for DNA library preparation (KAPA DNA library construction kit), and the 10%, 1%, and 0.1% samples after the step of ...

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Abstract

The invention discloses a double-label joint sequence for detection of tumor mutation, and is characterized in that the joint sequence is synthesized by a joint primer P5 and a joint primer P7, wherein the joint primer P5 has the sequence shown in SEQ ID NO:1, and the joint primer P7 has the sequence shown in SEQ ID NO:2. The invention also provides a database building method and a sequencing method. With use of a double-label joint, the tumor mutation rate of 1*10<-5> can be accurately detected, the tumor mutation detection sensitivity is effectively improved, and with combination of the flux of high-flux sequencing, one-time sequencing can detect multiple mutation loci of multiple genes.

Description

technical field [0001] The invention relates to the technical field of nucleic acid sequencing, in particular to a double-label linker sequence and a detection method for detecting tumor mutations. Background technique [0002] Due to the reasons of the sample preparation (library preparation) and the instrument system itself (oxidative damage or deamination damage of the DNA itself, mutations introduced by the PCR enzyme itself during the library construction process, and base reading by the instrument during sequencing), the current next-generation sequencing Errors introduced during the sequencing, etc.), the error probability of each base obtained by sequencing is between 1 / 1000-1 / 100, that is, there will be 1 to 10 error bases for every 1000 bases. [0003] In germline mutation (germline cell mutation) detection, the ratio of the mutation site in the sample is only 0%, 50% and 100%, so systematic base reading errors can be identified through data analysis. The overlapp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/10C40B50/06
CPCC12Q1/6806C12Q1/6869C12Q1/6886C12Q2600/156C40B50/06
Inventor 葛会娟林清华金保雷李旭超阮力
Owner AMOY DIAGNOSTICS CO LTD
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