Universal hybridization enhancer and method for targeted sequencing

A targeted sequencing and enhancer technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of unsmooth process, cumbersome operation, unfavorable automation platform establishment, etc., and achieve excellent blocking effect

Inactive Publication Date: 2020-11-20
伯科生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, 12 kinds of hybridization enhancers are required to perform hybridization capture on 12 libraries with 12 kinds of indexes, which is cumbersome to operate, the process is not smooth, and it is not conducive to the establishment of an automated platform

Method used

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  • Universal hybridization enhancer and method for targeted sequencing
  • Universal hybridization enhancer and method for targeted sequencing
  • Universal hybridization enhancer and method for targeted sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0038] 1. Pre-library construction

[0039] DNA library construction kit (Rapid DNA Lib Prep Kit, ABclonal) and Illumina TruseqDual-index adapter ( Figure 4 ), the pre-library used in this example was constructed on NA12878 gDNA (Coriell) (insert size: ~200bp; number of PCR cycles: 7).

[0040] 2. Hybrid capture

[0041] For B, A-D and C regions, design and synthesize LNA-containing oligonucleotides as components constituting universal hybridization enhancers, wherein oligonucleotides 3-UB 24LNA-B, 3-UB 24LNA-AD, 3- UB 24LNA-C is complementary to regions B, A-D and C, respectively, that is, blocks the above regions. Subsequently, using the specific hybridization enhancer corresponding to the Index sequence one-to-one as a control, hybridization capture was performed on the combination of universal blocking agent components, and the blocking effect was tested. A 4-hour hybridization capture (N=2) was performed following the steps indicated in A-J. The oligonucleotide seque...

Embodiment 2

[0103] This example is used to explore the Tm value of general hybridization enhancers. The method in Example 1 was used to construct the Illumina Truseq Dual-index linker pre-library of NA12878gDNA (Coriell).

[0104] As shown in Table 8, the specific hybridization enhancer and the general hybridization enhancer (1 nmoleEach) containing different amounts of LNA were used to carry out hybridization capture, sequencing and analysis of the pre-library (the same method as in Example 1) (N=2). See Table 1 for the sequence information of hybridization enhancers.

[0105] Table 8

[0106]

[0107] As shown in Table 9, as the Tm value increases, the On-Target ratio of the library gradually increases. When the Tm value of the universal hybridization enhancer reaches 80°C (3-UB 12LNA), its On-Target ratio is significantly improved compared with the On-Target ratio of the sample without the hybridization enhancer; when the Tm value of the universal hybridization enhancer reaches 83...

Embodiment 3

[0111] This example is used to study the blocking effect of universal hybridization enhancers on different Indexes.

[0112] Using the library construction method in Example 1, the pre-library used in this example was constructed on NA12878 gDNA (Coriell). Twelve cases of adapters were constructed with illumina Truseq Dual-index and pre-libraries containing different indexes (insert size: ~200bp; number of PCR cycles: 7). 3-UB 24LNA hybridization enhancer (1 nmole each) was used to carry out hybridization capture, sequencing and analysis of the above 12 pre-libraries (the method is the same as in Example 1).

[0113] As shown in Table 10, 8 kinds of Adapter-1 and 12 kinds of Index,3-UB 24LNA hybridization enhancers of Adapter-2 can obtain stable and efficient blocking effect.

[0114] Table 10

[0115]

[0116]

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Abstract

The invention discloses a universal hybridization enhancer and method for targeted sequencing. The hybridization enhancer provided by the invention can efficiently seal a linker sequence, is not influenced by Index in the linker sequence, is simple and convenient to operate and high in sealing efficiency, and can effectively reduce background signals, improve effective data volume and reduce sequencing cost.

Description

technical field [0001] The invention relates to the field of gene sequencing, in particular to a general hybridization enhancer for targeted sequencing and a targeted sequencing method. Background technique [0002] In the field of high-throughput sequencing, hybrid capture technology uses the principle of complementary pairing between nucleic acid molecules to efficiently enrich the target region of interest through biotin-modified probe molecules, thereby reducing the proportion of data in non-target regions and improving data quality. Efficient, it is a targeted sequencing technology with high practicability and low cost. At present, hybrid capture technology has been widely used in clinical diagnosis, such as accompanying diagnosis of tumor drugs, auxiliary diagnosis of genetic diseases, etc. [0003] For human gene-related hybridization capture, the hybridization reaction of probes and libraries (also known as pre-libraries) includes the following components, probes, l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6874
CPCC12Q1/6874
Inventor 卢亚明陈文浩韩营民
Owner 伯科生物科技有限公司
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