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Method for building sequencing library by hybridization

A library and genome library technology, applied in the field of second-generation sequencing technology, labeling technology, realizing sequence capture of multiple samples in the same reaction system, solexa sequencing technology, hybridization-based sequence capture technology, can solve the waste of sequencing data , poor sequence capture effect, non-target sequences, etc., to avoid data waste, avoid the reduction of sequencing length, and improve the efficiency of introduction

Active Publication Date: 2012-04-11
BGI TECH SOLUTIONS
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AI Technical Summary

Problems solved by technology

[0010] 1. The method of introducing tag sequences through adapters is not conducive to the application of this technology in sequencing platforms such as Solexa: on the one hand, the tag sequences added after adapter ligation are located between the sequencing primers and the sample DNA, and 11 bp must be sequenced before sequencing the sample DNA The method of sequentially sequencing the tag sequence and sample DNA with the same sequencing primer is used in the second-generation sequencing technology platform with a relatively short sequencing length, which will undoubtedly further shorten the effective sequencing length of the sample DNA; On the one hand, the introduction of tag sequences based on adapter ligation will cause both ends of the sample DNA to carry tag sequences, which will cause the tag sequences to be sequenced twice when sequencing platforms such as Solexa perform paired-end sequencing, resulting in a waste of sequencing data
[0011] 2. This technology does not use adapter blocks (also called blocking sequences), which will reduce the binding efficiency of the sample DNA and the probe due to the annealing between the adapter complementary sequences during the hybridization of the sample, affecting the sequence capture effect. At the same time, the sample DNA without any association may be connected due to the annealing between the adapters, and cascade amplification to form "macromolecular DNA". The target DNA is captured together, resulting in a large number of non-target sequences in the capture sequence
[0012] 3. Since this technology is mainly optimized for chip hybridization, when it is used in a liquid phase system, due to the small initial amount of sample DNA, they may have a greater impact on the sequence capture effect after they are connected together through adapters. Potentially captures a large number of off-target sequences
Therefore, the sequence capture effect of this technical solution on the Agilent liquid phase hybridization platform may be poor

Method used

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  • Method for building sequencing library by hybridization
  • Method for building sequencing library by hybridization
  • Method for building sequencing library by hybridization

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] The control example of embodiment 1.NimbleGen chip hybridization system (Roche NimbleGen company): Single sample is hybridized on Nimblegen 855K chip

[0083] (1) Experimental method:

[0084] 1. Hybrid library construction

[0085] Refer to the Illumina Multiplexing Sample Preparation Guide for the construction process of the hybridization library. After 3ug of genomic DNA (extracted from human peripheral blood) was interrupted, the ends were blunted, "A" bases were added, and adapters (from the Illumina Multiplexing Sample Preparation Oligonucleotide Kit) were added and carried out. PCR amplification, PCR reaction system and reaction conditions are as follows:

[0086] reaction system:

[0087]

[0088] Reaction conditions:

[0089] (a).98℃ 30s

[0090] (b).98℃ 15s

[0091] (c).65℃ 30s

[0092] (d).72℃ 30s

[0093] (e). Repeat steps (b)-(d) 3-9 times (4-10 cycles in total)

[0094] (f).72℃ for 5 minutes

[0095] (g). Stand at 4℃

[0096] Use Ampure beads ...

Embodiment 2

[0124] Example 2. Example of application in the NimbleGen chip hybridization system: 12 DNA libraries (constructed according to the hybridization library construction process) are mixed and then sequence captured with an 855K chip

[0125] (1) Experimental method:

[0126] 1. Preparation of hybrid library: the method is the same as that described in Example 1.

[0127] 2. Hybridization:

[0128] a. Sample preparation:

[0129]

[0130] The 12 libraries (constructed according to the hybridization library construction method) were mixed together and hybridized on the same chip, and the hybridization method was the same as in Example 1.

[0131] 3. Sequencing and data analysis: the method is the same as that described in Example 1.

[0132] (2) Results:

[0133] After the 12 samples were mixed, the effect of sequence capture with the 855K chip is shown in Table 2.

[0134] Table 2: Sequence capture effect with 855K chip after mixing 12 samples:

[0135]

Embodiment 3

[0136] Example 3. Example of application in the NimbleGen chip hybridization system: 24 libraries (constructed according to the hybridization library construction method) are mixed and then sequence captured with an 855K chip

[0137] (1) Experimental method:

[0138] 1. Preparation of hybrid library: the method is the same as in Example 1.

[0139] 2. Hybridization:

[0140] a. Sample preparation:

[0141]

[0142] 24 samples were mixed together and hybridized on the same chip, and the hybridization method was the same as in Example 1.

[0143] 3. Sequencing and data analysis: the method is the same as in Example 1.

[0144] (2) Results:

[0145] The effect of sequence capture with 855K chip after mixing 24 samples is shown in Table 3.

[0146] Table 3: Sequence capture effect with 855K chip after mixing 24 samples:

[0147]

[0148] Summary of the implementation results of the NimbleGen chip hybridization system: In Examples 2 and 3, 12 and 24 samples were respec...

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Abstract

The invention provides indexes for DNA sequencing, especially for a sequence acquisition technology, and a method for introducing the indexes by adopting a PCR (polymerase chain reaction) method. The invention also provides adapter block sequences for blocking adapters and application of the adapter block sequences in a NimbleGen chip hybridization system, an Agilent liquid hybridization system and a NimbleGen EZ liquid hybridization system. The invention further provides a method for building a library and / or sequencing by using the indexes and / or adapter block sequence, and the library built by the method.

Description

technical field [0001] The present invention relates to the technical field of nucleic acid sequencing, in particular to the field of sequence capture technology, especially the sequence capture technology based on hybridization. In addition, the present invention also relates to labeling technology and a method for realizing sequence capture of multiple samples in the same reaction system. The method of the present invention is particularly suitable for the second generation sequencing technology, especially the solexa sequencing technology. The methods of the invention are useful in genomics research. Background technique [0002] The second-generation sequencing technology represented by Illumina solexa, AB Solid and Roche 454 has greatly reduced the cost of sequencing, has developed rapidly in recent years, and has become an important tool for genomics research. Compared with the sanger sequencing technology of the chain termination method, the second generation sequen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C40B40/08C40B50/06
CPCC12N15/1093C12Q2563/179
Inventor 蒋慧刘晓吴仁花欧阳伟汉武靖华吴明枝赵美茹
Owner BGI TECH SOLUTIONS
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