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Label and preparation method and applications thereof

A tag and tag pool technology, applied in biochemical equipment and methods, DNA preparation, chemical library, etc., can solve the problems of high sequencing error rate, tag sequence change, misjudgment as another tag, etc., and achieve a high tag recognition rate , short test cycle, good stability

Active Publication Date: 2018-06-15
CAPITALBIO GENOMICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] For the use of tags, sequencing errors must be considered. If the sequencing errors happen to fall on the tag region, it will lead to changes in the tag sequence, making it impossible to distinguish the source of the sample, or misjudging it as another tag; in addition, as the number of tags used increases more, the higher the probability of label recognition error
For the Ion Torrent platform, its sequencing error rate is relatively high, so tags suitable for the Ion Torrent platform require a lower error tolerance rate, which will increase the difficulty of tag design and screening

Method used

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  • Label and preparation method and applications thereof
  • Label and preparation method and applications thereof
  • Label and preparation method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1, the method for preparing label

[0041] For the Ion torrent sequencing platform, the inventor provides a method for preparing tags, including the following steps:

[0042] (1) Generate all nucleotide sequences of 8bp and 10bp to form a pool of candidate tags.

[0043] (2) Screening the nucleotide sequences in the candidate tag pool, the screening criteria are as follows:

[0044] Remove the nucleotide sequence that is not C at the end of the 3' end;

[0045] Remove the nucleotide sequence whose 5' head is C;

[0046] Remove nucleotide sequences comprising AGC, GCC, AAG, CTGC, CCG, CAA, AAA, TTT, CCC, GGG;

[0047] Remove the nucleotide sequence starting from AGG at the 5' end;

[0048] Nucleotide sequences that are CC-terminated at the 3' end are removed.

[0049] (3) Perform pairwise alignment of the nucleotide sequences in the screened candidate tag pool, and remove any sequence with a difference of less than 3; use the Needleman-Wunsch algorithm fo...

Embodiment 2

[0052] Embodiment 2, label screening

[0053] Optionally select 415 tags in the candidate tag pool obtained in step (4) of Example 1, perform test screening, use the human genome as a template, and combine the deafness gene sequencing method (refer to Example 3 for specific methods) to carry out library construction, and the 415 library High-throughput sequencing was carried out on a PI chip, and the amplification efficiency of the tag sequence was measured according to the amount of data obtained by sequencing (number of reads). The ratio of the amount of sequencing data to the average amount of data was greater than 25% as the standard, and a total of 384 samples were screened. tag sequences, and arrange the sequencing data volume in descending order, as shown in Table 1.

[0054] Table 1. Tag screening

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

Embodiment 3

[0066] Example 3. Deafness gene sequencing method based on DNA tag library

[0067] 1. Tab connector

[0068] Using the 384 tags (hereinafter referred to as index) shown in SEQ ID NO: 004 to SEQ ID NO: 387 shown in Table 1, insert the tag upstream of the 3' end "GAT" of the tag adapter, and the tag adapter 3' end "GAT" "The universal sequence is inserted downstream, wherein the original tag linker sequence is 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAGGAT-3' (SEQ ID NO: 001), and the universal sequence is: 5'-AAATGGGCGGTAGGCTTG-3' (SEQ ID NO: 002); thus constituted The new 384-index linker has the following structural form: 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG-index-GAT-AAATGGGCGGTAGGCTTG-3'.

[0069] 2. DNA extraction

[0070] A total of 1152 dried blood spot samples (400 positive samples and 752 negative samples) were selected, and the samples were scientific research samples provided by cooperative units. They were extracted using HiPure Blood DNA Mini Kit (Magen Company), and the pur...

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Abstract

The invention discloses a label preparation method, a label, a label connector, and a gene sequencing method based on a DNA label library. The provided label preparation method can rapidly obtain a label sequence. The label screening and testing period is short. 384 labels and label connectors obtained by the method can be applied to DNA label library building and gene sequencing, sequencing on 384 samples can be carried out parallelly, the stability is good, the label recognition rate and the accuracy are high, and the label is especially suitable for an Ion torrent platform.

Description

technical field [0001] The invention belongs to the field of gene sequencing, and in particular relates to a method for preparing a tag, a tag, a tag adapter and a gene sequencing method based on a DNA tag library. Background technique [0002] High-throughput sequencing technology is a revolutionary change to traditional next-generation sequencing, which can sequence hundreds of thousands to millions of nucleic acid molecules at a time. One of the advantages of high-throughput sequencing over next-generation sequencing is massively parallel sequencing. The key to achieving massively parallel sequencing lies in the application of tags, which can be used to mark nucleic acids from different sample sources, so as to achieve parallel detection of a large number of samples, which greatly shortens the total sequencing time. [0003] For the use of tags, sequencing errors must be considered. If the sequencing errors happen to fall on the tag region, it will lead to changes in the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869C12N15/10C12N15/11C40B50/06C12Q1/6883C12Q1/6886
CPCC12N15/1013C12N15/1065C12Q1/6869C12Q1/6883C12Q1/6886C40B50/06C12Q2525/191C12Q2535/122
Inventor 糜庆丰朱鹏远吴春求黄铨飞潘兆东王杨周幸芝刘丽菲
Owner CAPITALBIO GENOMICS
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