Sequencing method for in-situ copying high-flux sequencing template and increasing reading length thereof

A high-throughput, sequencing technology, applied in the field of high-throughput sequencing, can solve the problems affecting the efficiency of splicing and assembly, reducing the read length of the sequence, and increasing the error of sequencing, so as to reduce the number of repeated determinations, increase the read length, and improve the accuracy sexual effect

Inactive Publication Date: 2013-08-07
SOUTHEAST UNIV
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  • Claims
  • Application Information

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Problems solved by technology

In these sequencing-by-synthesis methods, whether it is pyrosequencing, extension sequencing of labeled monomers, or ligation sequencing, as the number of extension (or ligation) reactions increases, due to its elongation (or ligation) efficiency, cleavage efficiency, sequencing Due to the loss of primers and other effects, sequencing errors will continue to increase, resulting in a decrease in the sequence read length, and the sequence read length significantly affects the efficiency of splicing and assembly

Method used

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  • Sequencing method for in-situ copying high-flux sequencing template and increasing reading length thereof
  • Sequencing method for in-situ copying high-flux sequencing template and increasing reading length thereof
  • Sequencing method for in-situ copying high-flux sequencing template and increasing reading length thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Example 1: Regeneration of high-throughput sequencing templates and its ligation sequencing method to determine the genome of Escherichia coli

[0024] (1) Put ~100ng / mL Escherichia coli genome sample 100mL on the ultrasonic instrument for 20 minutes at low frequency, and the ultrasonic results were detected by electrophoresis (such as image 3 ), and recover DNA fragments with a length of 100±30bp from the gel.

[0025] (2) Ligate the linker 1, 2 (see the table below for the specific sequence) and the above 100±30bp fragmented nucleic acid sequence under the action of ligase (the two ends of all different template molecules contain the same sequence, that is, the linker) .

[0026]

[0027] (3) The biotin-modified amplification primer and extension primer (see the table below for the specific sequence) fully react with the avidin-modified magnetic microspheres to immobilize them on the magnetic microspheres.

[0028] Primer sequence template amp...

Embodiment 2

[0037] Example 2: Regeneration of high-throughput sequencing templates and its ligation sequencing method to determine the human genome

[0038] (1) The magnetic microsphere human genome sequencing template chip was prepared according to the method in Example 1 (see steps (1) to (4) in Example 1).

[0039] (2) Install the glass slide with fixed microsphere human genome sequencing template into a high-throughput sequencer to construct a reaction pool, and follow the extended sequencing method (Bentley, D. R. et al . Accurate whole human genome sequencing using reversible terminator chemistry. Nature, 2008, 456, 53-59) program to obtain the fluorescent signal of each reaction of each magnetic bead and convert the signal into base information ( Figure 5 ), when performing 35 times of extended sequencing to determine the 35 base sequence information of the template.

[0040] (3) Add 0.1M NaOH solution into the reaction pool to denature the sequencing product into single-stran...

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Abstract

The invention relates to a sequencing method for in-situ copying high-flux sequencing template and increasing reading length thereof, which comprises the following steps of: after obtaining a sequence fragment by sequencing a prepared DNA (Deoxyribonucleic Acid) sequencing template, denaturing the DNA sequencing template to DNA single chains--old templates; copying the template by activating a previously introduced extending primer and then fully cutting off the old template to obtain DNA single chains--new templates which completely complement the original DNA sequencing template; carrying out sequence measurement by using the DNA single chains as DNA sequencing templates to obtain new measured sequences complementary with the other ends of the old templates. The sequence fragments obtained by measuring the new templates and the old templates are spliced so that the reading length of the sequencing template is increased, the difficulty of splicing short fragment sequences is reduced,and the accuracy of sequences is improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for increasing the length of sequencing reads in DNA sequence analysis, in particular to a high-throughput sequencing method for increasing the length of reads through in-situ replication of sequenced templates. Background technique [0002] With the development and completion of the Human Genome Project and various model organism genome projects, human beings have entered the post-gene era, which has had a huge impact on contemporary biological research and medical research, and the related disciplines of molecular biology have been rapidly developed. develop. It will become possible to understand the differences of life, the law of disease occurrence and development, and the interaction between drugs and living organisms at the genetic level. As far as gene sequence analysis is concerned, the focus of the post-genome era has shifted from whole-genome sequence determinatio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 肖鹏峰陈婧葛芹玉陆祖宏
Owner SOUTHEAST UNIV
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