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Method for constructing DNA large fragment library and application thereof

A construction method and large fragment technology, applied in the field of molecular biology, can solve the problems of short read length, inability to evaluate library quality, time cost, personnel cost and instrument cost waste, etc.

Active Publication Date: 2018-03-13
ZHEJIANG ANNOROAD BIO TECH CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of next-generation sequencing is the short read length. The Roche 454, which has the longest read length in the second-generation sequencer, can only perform DNA sequencing of up to 400 bp. According to the overlapping sequences on different small fragments, their sequencing structures are spliced ​​into contigs of different sizes (fragment contigs)
The existing DNA large fragment library construction methods cannot evaluate the quality of the library during the process of building the library or before sequencing, and it is necessary to judge whether the library construction is successful or not based on the sequencing results after the sequencing is completed.
High-throughput sequencing of unqualified libraries will result in a lot of waste of time, personnel and instrument costs. Being able to evaluate the quality of large-fragment libraries during library construction or before sequencing has become a major issue in large-fragment library construction and even denovo. Sequencing technology is urgently needed to solve the problem

Method used

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  • Method for constructing DNA large fragment library and application thereof
  • Method for constructing DNA large fragment library and application thereof
  • Method for constructing DNA large fragment library and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] Embodiment 1 large fragment DNA library construction

[0116] 1. Large fragment processing of DNA samples

[0117] 1.1 Take 1 mL of healthy human whole blood and extract it according to the operating instructions of the blood genome DNA extraction system (0.1-20 mL) (Tiangen Biochemical Technology Co., Ltd.) to obtain DNA samples. Take 5 μg of DNA sample, and use the HydroShear Plus DNA Fragmentation Instrument to fragment large fragments of DNA samples according to the instructions. The target large fragment length is 5kb.

[0118] 2. End repair

[0119] 2.1 Prepare the end repair reaction system in a 1.5mL centrifuge tube:

[0120]

[0121] dNTP Mix (dNTP Mixture) (10mM each) is a premix solution containing sodium salts of dATP, dCTP, dGTP and dTTP, each concentration is 10mM, the total concentration is 40mM (pH7.5).

[0122] 2.2 Incubate at 20° C. for 30 minutes in a Thermomixer C constant temperature mixer (EPPENDORF company, hereinafter referred to as thermo...

Embodiment 2

[0201] Embodiment 2 carries out quality evaluation to the library that embodiment 1 obtains

[0202] 1.1 The large fragment library product obtained in Example 1 was digested with restriction endonuclease HindIII.

[0203] Prepare the enzyme digestion reaction system according to the following table:

[0204]

[0205] 1.2 Place in a thermostat at 37°C for 2 hours. DNA was recovered by gel purification using an agarose gel recovery kit.

[0206] 1.3 The library product of Example 1 and the result product of step 1.2 were detected by electrophoresis using 2% agarose gel, and the results were as follows: figure 1 shown.

[0207] Judgment criteria: When the average value of the electrophoresis bands after digestion of the library moves to about half of the average size of the original library bands, and presents a more dispersed state than the library bands, the library construction is successful.

[0208] Depend on figure 1 It can be seen that the average efficiency of li...

Embodiment 3

[0209] Example 3 The library obtained in Example 1 is carried out on-machine sequencing and analysis of sequencing results

[0210] 1.1 Run the paired-end sequencing program (PE150) on the HiSeq 2500 sequencing platform for the large-fragment sequencing library obtained in step 13.3, and obtain off-machine data as shown in Table 1.

[0211] Table 1

[0212]

[0213] It can be seen from Table 1 that the ratio of Clean reads to Raw reads is higher, Q30 is higher, and the overall sequencing results are better.

[0214] 1.2 The sequence reads of 100 bp were intercepted for analysis, and the linker sequence of Example 1 was removed by the software DeLoxer (non-patent literature 2) to remove the loxp linker, and the Clean reads data was classified. The classification results are shown in Table 2.

[0215] Table 2:

[0216]

[0217] Mate-paired data is the value and ratio of the real paired-end fragments in the large fragment library processed by DeLoxer software, which is ef...

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Abstract

The invention relates to a method for constructing a DNA large fragment library and an application thereof. The method is used for construction of a DNA large fragment library. The method comprises introducing a cyclizing DNA large fragment to a cleavage site. Through the method, a library with a quality control site or a library intermediate product can be obtained so that the quality evaluationof the library before sequencing can be realized.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a method for constructing a DNA large fragment library, a DNA large fragment library constructed by the method and an application of the DNA large fragment library in sequencing. Background technique [0002] For a species whose genome sequence is unknown or has no genome information of related species, its genome DNA fragments of different lengths and their libraries are sequenced, and then spliced, assembled and annotated by bioinformatics methods to obtain the complete genome of the species Sequence map, called genome de novo sequencing, also called de novo sequencing. Today, with the rapid development of genomics, the combination of de novo sequencing and comparative genomic methods can be used to explore the origin and evolution of the species, and to study the molecular mechanisms of its growth and development, shape production and environmental adaptation, whic...

Claims

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Application Information

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IPC IPC(8): C40B50/06C40B40/06C12Q1/6869
CPCC12N15/1093C12Q1/6869C40B40/06C40B50/06C12Q2525/191C12Q2525/131C12Q2535/122
Inventor 梁峻彬李小林张介中韩典昂刘三阳玄兆伶李大为陈重建
Owner ZHEJIANG ANNOROAD BIO TECH CO LTD
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