Method for preparing circular ssDNA and kit of method

A kit and circular technology, applied in a method for preparing circular ssDNA and its kit, in the field of next-generation sequencing, can solve the problems of poor sequencing uniformity of high GC content samples, damaged library DNA strands, etc., and achieve improved sequencing Uniformity, the effect of improving the quality of sample sequencing

Pending Publication Date: 2020-12-11
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The higher the GC content of the library, the higher the temperature required for denaturation; but if the temperature is too high, the DNA strand of the library is easily damaged
This results in poor sequencing uniformity for high GC content samples with this ssDNA preparation method

Method used

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  • Method for preparing circular ssDNA and kit of method
  • Method for preparing circular ssDNA and kit of method
  • Method for preparing circular ssDNA and kit of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Preparation of Halobacterium halophilus (GC%=64) circular ssDNA by the method of the present invention

[0032] 1. Synthesis of amplification primers:

[0033] According to the method for preparing circular ssDNA of the present invention, primers were synthesized, and primer Ad153_PCR 2-2 was subjected to 5' phos modification to obtain the primer pair as follows:

[0034] Ad153_PCR 2-1: GAACGACATGGCTACGA (SEQ No. 1)

[0035] Ad153_PCR 2-2: 5'-p-TGTGAGCCAAGGAGTTG (SEQ No.2)

[0036] 2. Construction of Halobacterium library (MGI platform):

[0037] A, sample selection: in this example, select halophilic bacilli with GC content of 64%;

[0038] B. DNA fragmentation: the sample gDNA is interrupted, and the fragmentation method selected in this example is the mechanical method;

[0039] C. Library construction: using Ultima DNA Library Prep Kit for formula kit, please refer to the instruction manual of the library construction kit for details; the amplifica...

Embodiment 2

[0056] Example 2 High-throughput sequencing and result analysis

[0057] In this example, we used the halophilic library as a template, prepared single-stranded circular ssDNA by the above two circularization methods, and sent it to the MGI sequencing platform for sequencing in the same lane of the same sequencer. The sequencer is BGISEQ-2000, mode PE100, the amount of sequencing data is 1G; the quality of sequencing data is shown in Table 1 and Table 2.

[0058] Table 1 Different circularized ssDNA preparation methods - comparison of sequencing quality 1

[0059]

[0060] Table 2 Different circularized ssDNA preparation methods - comparison of sequencing quality 2

[0061]

[0062] The results show that the ssDNA library prepared by the method of the present invention has one of the important indicators for measuring the sequencing quality - the base quality value is higher than that of the ssDNA library prepared by the traditional method, and its Q20 and Q30 are incre...

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Abstract

The invention discloses a method for preparing circular ssDNA and a kit of the method. The method comprises the following steps: 1) breaking sample gDNA; 2) constructing a DNA library, wherein one ofthe amplification primers is modified with 5 '-phosphoric acid; 3) performing enzyme digestion on one chain modified with 5 '-phosphoric acid in double chains of the library; 4) performing 5 'phosphorylation on the other chain; and 5) performing cyclizing by ligase and splint oligo. The kit comprises exonuclease, 5'-terminal phosphorylase, ligase and splint oligo suitable for double-chain DNA of which the substrate is 5' phosphorylation. The method has the advantages that formation of circular dsDNA can be avoided, the sequencing quality of samples is improved, and the sequencing uniformity ofsamples with high GC content is improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, relates to next-generation sequencing technology, in particular to a method for preparing circular ssDNA and a kit thereof. Background technique [0002] Next generation sequencing technology (next generation sequencing, NGS) is a revolutionary change to generation sequencing technology, which has the characteristics of high throughput and low cost (single base). The emergence of high-throughput sequencing technology has directly reduced the cost of human genome sequencing by 5,000 times, providing a major boost to the smooth progress of the Human Genome Project. NGS technology is constantly improving, and the amount of sequencing data has also increased by 100-1000 times, but due to its own characteristics, the accuracy is not as good as that of first-generation sequencing. [0003] In order to effectively improve the sequencing accuracy, the BGISEQ sequencing platform adopts DNBSEQ TM technology....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12N15/11
CPCC12Q1/6806C12Q2525/113C12Q2521/501C12Q2521/325C12Q2531/113C12Q2535/122
Inventor 李真真王家庭曹振宋东亮周其好
Owner YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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