Y-shaped linker, kit and method for constructing RRBS library

A kit and library technology, applied in the field of molecular breeding, can solve problems such as low enzyme digestion efficiency evaluation results, achieve the effects of improving its accuracy, avoiding mismatching, and improving accuracy

Pending Publication Date: 2020-11-17
天津诺禾医学检验所有限公司
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The main purpose of the present invention is to provide a Y-shaped linker, a kit and a method for constructing an RRBS library, so as to solve the defect of low enzyme cutting efficiency evaluation results in the prior art

Method used

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  • Y-shaped linker, kit and method for constructing RRBS library
  • Y-shaped linker, kit and method for constructing RRBS library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] (1) Sampling

[0045] According to the library building task list, carefully check whether the Novo number, sample name, and sample status of the sample are consistent with those marked on the library building list, place the samples in order, shake briefly after all the samples are melted, and centrifuge for sampling.

[0046] Prepare a 200 μL EP tube according to the library construction task, and add the corresponding volume of RNase-free H 2 O, according to the sampling volume of the library construction task list, take samples in sequence (3 μg genomic DNA for each sample), check the sample name and Novo number again when sampling, and take the sample after confirming that it is correct. After the sample is taken, the EP tube and the sample tube are one by one Correspondingly put it in the ice box, and continue to take the next sample. After all the samples are taken, check whether the sequence of the sample tubes is consistent with the sequence of the EP tubes. Af...

Embodiment 2

[0170] In this example, the same sample to be tested as in Example 1 was used, and the library was constructed according to the same steps, wherein the only difference was the Y-shaped joint, and the specific sequence information was as follows:

[0171] The first group: Compared with SEQ ID NO: 3, the methylated adapter 1 only lacks the base t complementary to the sequencing primer; compared with SEQ ID NO: 4, the methylated adapter 2 only lacks the base t complementary to the sequencing primer The same base a in the end base.

[0172] The second group: compared with SEQ ID NO: 3, the protected base sequence of methylated linker 1 is atg (reduced by half), compared with methylated linker 2 and SEQ ID NO: 4, the protected base sequence is tac (cut in half).

[0173] The third group: compared with SEQ ID NO: 3, methylated linker 1 has three tacs added at the end of the protected base sequence.

[0174] The fourth group: the methylated linker 1 is the same as SEQ ID NO: 3, and...

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Abstract

The invention provides a Y-shaped linker, a kit and a method for constructing an RRBS library. The Y-shaped linker comprises a methylation linker 1, namely 5'-acactctttccctacacgacgctcttccgatctn-3', and a methylation linker 2, namely 5'-n2n1' agatcggaagagcacacgtctgaactccagtcac-3', wherein the methylation linker 1 and the methylation linker 2 are annealed to form the Y-shaped linker; the bases c inthe methylation linker 1 and the methylation linker 2 are methylationmodified c; n1 and n1' are complementary paired protective base sequences; the number of bases of n1 or n1' is in a range of 6-12;n2 is a cohesive terminal base of an enzyme cutting site; and the number of bases of n2 is in a range of 1-5. The linker is helpful for improving sequencing data output, and also improves the evaluation and accuracy of enzyme digestion efficiency.

Description

technical field [0001] The invention relates to the field of molecular breeding, in particular to a Y-shaped linker, a kit and a method for constructing an RRBS library. Background technique [0002] RRBS (Reduced representation bisulfite sequencing), that is, degenerate representative bisulfite sequencing technology, is an efficient high-throughput sequencing technology for the analysis of methylation level at the single nucleotide level of the genome. The principle is to use the combination of restriction endonuclease MspI and fragment selection to enrich the high GC region in the genome, and then use the bisulfite sequencing method to obtain the methylation level analysis results at the single nucleotide level of the genome. [0003] The current library construction scheme consists of the following steps: enzyme digestion --- purification --- end repair plus A tail --- ligation of adapters --- CT conversion --- recovery of the target fragment. However, after obtaining th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12N15/11C12Q1/6806
CPCC40B50/06C12Q1/6806C12Q2525/191C12Q2525/117
Inventor 郭松孔维茜李彪骞蕾阳吴国营沈世超
Owner 天津诺禾医学检验所有限公司
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