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Universal closed sequence and its applications

A technology of blocking sequences and tagging sequences, which is applied in the field of high-throughput sequencing library construction, can solve the problems of low hybrid capture efficiency of paired-end index libraries, and achieve the effects of improving capture rate, avoiding capture, and improving sealing effect

Active Publication Date: 2021-07-23
NANODIGMBIO (NANJING) BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The main purpose of the present invention is to provide a universal blocking sequence and its application to solve the problem of low hybrid capture efficiency of double-ended index libraries in the prior art

Method used

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  • Universal closed sequence and its applications
  • Universal closed sequence and its applications
  • Universal closed sequence and its applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0140] The steps of Example 2 are the same as those of Example 1, the only difference is that the number of modified bases of the universal blocking sequence used is different. In this embodiment, the number of modified bases of the universal closed sequence is shown in the table below:

[0141] Table 8:

[0142] closed combination 4+6 modification blocking (blocking scheme 3) 8+11 modification combination (blocking scheme 4) Concentration (μmol / L) 200 100

[0143] In the blocking scheme 3, the P1 sequence remains unchanged (the P1 end blocking sequence shown in SEQ ID NO:3,

[0144] CTCTCA+GTACG+TCA+GCA+GT+T10XXXXXXXXXXCA+ACTCCT+TGGC+TCACAGA+ACGA+CATGG+CTACGATC+CGACTT / 3SpC3 / ), P2 sequence is: SEQ ID NO:7

[0145] GCA+TGGC+GA+CCTT+ATCAGXXXXXXXXXXTTGTCTT+CCTA+AGA+CCGC+TTG+GCCTCCGA+CTT / 3SpC3 / .

[0146] In the blocking scheme 4, the P1 blocking sequence is SEQ ID NO:8

[0147] CTC+TCA+GT+ACG+TCA+G+CA+GT+TXXXXXXXXXCA+ACTCCT+TGGC+TCAC+AGA+ACGA+CAT+GG+CT...

Embodiment 3

[0151] The steps of Example 3 are the same as in Example 1, and the number of modified bases of the universal closed sequence is also the same as that of Scheme 2 of Example 1. The only difference is that the positions of the modified bases of the universal closed sequence are different, and the specific sequence is as follows:

[0152] Blocking Scheme 5: The P1 blocking sequence is SEQ ID NO: 10

[0153] CTC+T+CA+GT+ACG+TCA+GCA+GTTXXXXXXXXXXCAACTCCT+TGGC+TCAC+AGA+ACGA+CAT+GG+CTAC+GATC+CGA+CTT / 3SpC3 / , the closed sequence of P2 is SEQ ID NO: 11

[0154] G+CA+TG+GC+GA+CC+TT+ATCAGXXXXXXXXXXTTGTCTT+CCTA+AGA+CC+GC+TTG+GCC+TC+C+GA+CTT / 3SpC3 / .

[0155] Blocking Scheme 6: The P1 blocking sequence is SEQ ID NO: 12

[0156] CTCTCA+GT+ACG+TCA+G+CA+GT+TXXXXXXXXXCA+ACT+CCT+TGGC+TCAC+AGA+ACGA+CAT+GG+CTAC+GATCCGACTT / 3SpC3 / ; P2 closed sequence is SEQ ID NO: 13

[0157]GCATG+GC+GA+CC+TT+AT+CA+GXXXXXXXXXXTTG+TCTT+C+CTA+AGA+CC+GC+TTG+GCC+TCC+GACTT / 3SpC3 / .

[0158] Closing scheme 5 is to chang...

Embodiment 4

[0159] Example 4 Multiple library hybridization test

[0160] The experimental steps of library construction and hybridization are the same as in Example 1. The base modification of 7+10 is selected for universal blocking, and the concentration is 100 μmol / L. The difference is that multiple libraries are mixed and hybridized. The specific number of libraries input and the total amount of library inputs are as follows:

[0161] Table 7.

[0162] Input library quantity (500ng / library) 10 12 14 16 Total library input 5μg 6μg 7μg 8μg

[0163] When the input amount of hybridization capture is 500ng / library, the performance of sequencing indicators is better. If a single hybridization can allow more input, that is, more samples in a single hybridization will reduce the hybridization capture cost of each sample. . The test of the universal closed sequence of this application found that when no more than 12 samples were hybridized together in a sin...

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Abstract

The invention provides a universal closed sequence and its application. According to the direction from 5' to 3', the general closure sequence includes the left non-tag region closure sequence, the middle label region closure sequence and the right non-tag region closure sequence connected sequentially, and the left non-tag region closure sequence includes 5-7 bases modified by LAN or BNA, the closed sequence of the label region in the middle is a universal closed base sequence, the closed sequence of the non-label region on the right includes 7-10 bases modified by LAN or BNA, and the closed sequence of the non-label region on the right The 3' end has a blocked modification. By designing 5-7 base sequences and 7-10 base sequences modified by LNA or BNA in the left and right non-label regions, the binding ability to the sequence to be blocked can be significantly enhanced, thereby increasing the blocking effect; the 3' end is blocked The modification can reduce or avoid the non-specific capture of the library and improve the on-target rate of the target library capture.

Description

technical field [0001] The invention relates to the field of high-throughput sequencing library construction, in particular to a universal closed sequence and its application. Background technique [0002] With the increasing importance of high-throughput sequencing in auxiliary diagnosis of clinical applications, how to reduce the cost of sequencing is a key issue, and the reduction of sequencing cost has corresponding performance at different levels: MGI has continuously launched higher Sequencing throughput of sequencers, the cost of sequencing has been continuously reduced, and MGI-200, MGI-2000 and T7 sequencers have been launched successively, among which the T7 sequencer is currently the sequencer with the highest sequencing throughput and the lowest sequencing cost on the market. In addition, targeted capture sequencing is also an effective way to achieve large-scale reduction of detection costs while detecting target sequences. [0003] In the process of sequencing...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C40B50/06C12Q1/6806
CPCC12N15/113C12N2310/10C12Q1/6806C40B50/06C12Q2531/113
Inventor 胡玉刚汪彪郑文莉吴强
Owner NANODIGMBIO (NANJING) BIOTECHNOLOGY CO LTD
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