Nucleic acid sample content determining method for genome next generation sequencing

A technology of next-generation sequencing and determination methods, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve problems such as inaccurate quantitative results, improve quantitative accuracy, reduce the amount of standard substances, and reduce quantitative costs. Effect

Inactive Publication Date: 2019-06-11
武汉至纯科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In view of the above defects or improvement needs of the prior art, the present invention provides a nucleic acid sample content determination method for genome next-generation sequencing, the purpose of which is to use a more accurate standard curve to replace the standard that may not be effective in the current experiment. Curve, thereby solving the technical problem of inaccurate quantitative results of the existing nucleic acid sample content determination method for gene next-generation sequencing

Method used

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  • Nucleic acid sample content determining method for genome next generation sequencing
  • Nucleic acid sample content determining method for genome next generation sequencing
  • Nucleic acid sample content determining method for genome next generation sequencing

Examples

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Embodiment 1

[0066] A method for determining the content of a nucleic acid sample for next-generation sequencing of a genome, comprising the following steps: performing a fluorescent quantitative polymerase chain reaction (Qpcr) on a standard substance with a concentration of 20 pM and three nucleic acid samples to be tested to obtain a given fluorescence intensity The number of cycles of the next standard and the nucleic acid sample to be tested; the results are as follows:

[0067]

[0068] (2) According to the number of cycles of the standard substance obtained in step (1), select an applicable standard curve from the standard curve series;

[0069] Described standard curve embodies the linear relationship between the logarithm of initial concentration and the number of cycles; the point that embodies the given concentration logarithm value of standard substance and the number of cycles thereof in step (1) is as follows: figure 1 The point P shown in , according to condition 1, selec...

Embodiment 2

[0084] A method for determining the content of a nucleic acid sample for next-generation sequencing of a genome, comprising the following steps:

[0085] (1) Perform fluorescence quantitative polymerase chain reaction (Qpcr) on 4 standard substances with concentrations of 20pM, 2pM, 0.2pM, 0.02pM and 3 nucleic acid samples to be tested to obtain the standard substance and nucleic acid samples to be tested at a given fluorescence intensity The number of cycles; the results are as follows:

[0086]

[0087] (2) According to the number of cycles of the standard substance obtained in step (1), select an applicable standard curve from the standard curve series;

[0088] The standard curve embodies the linear relationship between the logarithm of the initial concentration and the number of cycles;

[0089] The points that reflect the logarithmic value of the given concentration of the standard in step (1) and its cycle times are as follows: figure 2 Point P shown in 1 ,P 2 ,P ...

Embodiment 3

[0097] A method for determining the content of a nucleic acid sample for next-generation sequencing of a genome, comprising the following steps:

[0098] (1) Perform fluorescence quantitative polymerase chain reaction (Qpcr) on a standard substance with a concentration of 20pM, 7 nucleic acid samples to be tested, and a quantitative reference substance to obtain the circulation of the standard substance and the nucleic acid sample to be tested at a given fluorescence intensity times; the results are as follows:

[0099]

[0100] (2) According to the number of cycles of the standard substance obtained in step (1), select an applicable standard curve from the standard curve series;

[0101] The standard curve embodies the linear relationship between the logarithm of the initial concentration and the number of cycles;

[0102] The points that reflect the logarithmic value of the given concentration of the standard in step (1) and its cycle times are as follows: image 3 Poin...

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Abstract

The invention discloses a nucleic acid sample content determining method for genome next generation sequencing. The method comprises the following steps of (1) performing a fluorescent quantitative polymerase chain reaction on one or more quantitative-concentration standard product and nucleic acid samples to be tested to obtain the cycle number of the standard products under the quantitative fluorescent intensity and the nucleic acid samples to be tested; (2) according to the cycle number of the standard products obtained in the step (1), selecting an appropriate standard curve in a standardcurve series; and (3) according to the cycle number of the nucleic acid to be tested, obtained in the step (1) and the standard curve selected in the step (2), demarcating the concentration of the nucleic acid samples to be tested. An accurate standard curve of which the test condition is consistent with that of the current test, and which is accurate is selected, so that the quantifying error canbe notably reduced, and the quantifying accuracy can be improved. Besides, a preferred plan can notably reduce the consumption of the standard product, the material consumption can be saved, and thequantifying cost can be reduced.

Description

technical field [0001] The invention belongs to the technical field of genome sequencing, and more specifically relates to a nucleic acid sample content determination method for genome next-generation sequencing. Background technique [0002] With the rapid development of second-generation sequencing technology, the scientific community has begun to apply more and more second-generation sequencing technology to solve biological problems. The application of second-generation sequencing technology platforms, including Roche's 454 sequencing platform, ABI's Solid sequencing platform, and Illumina's Solexa sequencing platform, have had a major impact on metagenomic research. Among them, Illumina's Solexa and Hiseq should be said to be the most widely used second-generation sequencing machines in the world. With the increasing application of next-generation sequencing, the problem of sequencing library quantification has become prominent: if the quantification of the library is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869
Inventor 陈钏侯玉彬向浩张露露
Owner 武汉至纯科技有限公司
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