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Label joint for detection of ultra-low-frequency gene mutation and application of label joint
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A technology of tagging and sequencing, applied in the field of sequencing, can solve the problems such as the need to improve the tag connector and the low sensitivity of sample mutation detection, and achieve the effects of easy implementation, simple operation and accurate detection results.
Active Publication Date: 2016-10-26
GENETALKS BIO TECH CHANGSHA CO LTD
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[0002] In the current stage of library construction and sequencing, the tested samples are connected with index adapters, which can distinguish samples from different sources, but the detection sensitivity of sample mutations is low
[0003] Therefore, the current label joints still need to be improved
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Embodiment 1
[0059] The library construction and the L858R mutation detection of exon 21 of the EGFR gene were performed on the sample using the tag adapter of the present invention (with the illumina Truseq adapter as the control), as follows:
[0060] 1. Label joint design of the present invention
[0061] Prelib-ADT1-S: CAAGCAGAAGACGGCATACGAGATTNNNNNNNNggaattaGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO: 1),
[0062] Prelib-ADT2-S: CAAGCAGAAGACGGCATACGAGATNNNNNNNNatccggcGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO: 2),
[0063] Prelib-ADT3-S: CAAGCAGAAGACGGCATACGAGATNNNNNNNNNcaggccgGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO: 3),
[0064] Prelib-ADT-AS: pGATCGGAAGAGC,
[0065] The 5' end of the Prelib-ADT-AS sequence is modified with a phosphate group.
[0066] Prelib-ADT1-S, Prelib-ADT2-S, Prelib-ADT3-S (both are the first strands of the joint) are annealed with the Prelib-ADT-AS sequence to form double strands respectively, forming three joints of the present invention Prelib-A...
Embodiment 2
[0178] Using the tag adapter of the present invention (using the existing single-molecule tag-containing adapter as a control) to perform library construction and KRAS gene exon 2 G12D point mutation detection, the details are as follows:
[0179] 1. Label joint design of the present invention
[0180] Prelib-ADT1-S: CAAGCAGAAGACGGCATACGAGATTNNNNNNNNggaattaGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO: 1),
[0181] Prelib-ADT-AS: pGATCGGAAGAGC, which is the modification of the phosphate group at the 5' end of the Prelib-ADT-AS sequence.
[0182] Prelib-ADT1-S and Prelib-ADT-AS sequences are annealed into double strands to form the linker Prelib-ADT1 of the present invention.
[0183] 2. The control group contains random single-molecule tag adapter design
[0184] According to the method described in the following literature, the design and manufacture of adapters containing single molecule tags: Scott R Kennedy, Michael W Schmitt.Detecting ultralow-frequency mutations by Dup...
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Abstract
The invention discloses a label joint for detection of ultra-low-frequency genemutation and an application of the label joint. The label joint comprises a first chain and a second chain, wherein the first chain comprises a first nucleic acid sequence, a second nucleic acid sequence, a third nucleic acid sequence and a fourth nucleic acid sequence from the 5' end to the 3' end sequentially; the first nucleic acid sequence is a universal primer binding target region; the second nucleic acid sequence is a random single-molecular label sequence and used for distinguishing DNA fragments; the third nucleic acid sequence is a sample label sequence and used for distinguishing library samples; the fourth nucleic acid sequence is a sequencing primer binding target region; the second chain is a sequence reversely complementary to the 3' end of the first chain to form double-strand. The label joint is introduced into a tested sample, can effectively improve the mutation detection sensitivity of the tested sample and can even detect mutation sites with mutation frequency as low as 0.1%; besides, the label joint is easy to prepare and has quite high actual application values.
Description
technical field [0001] The invention relates to the technical field of sequencing, in particular to a tag adapter for detecting ultra-low frequencygene mutations and an application thereof. Background technique [0002] In the current stage of library construction and sequencing, the tested samples are connected with index adapters, which can distinguish samples from different sources, but the detection sensitivity for sample mutations is low. [0003] Therefore, the current tag joints still need to be improved. Contents of the invention [0004] The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, an object of the present invention is to propose a label linker that can improve the detection sensitivity of low-frequency mutations in samples. [0005] It should be noted that the present invention is based on the inventor's following discoveries and work: [0006] In the process of high-throughput sequencing, th...
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