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Method of correcting amplification deviation in amplicon sequencing

An amplicon and bias technology, which is used in biochemical equipment and methods, sequence analysis, and microbial determination/inspection. Problems such as increasing deviation

Active Publication Date: 2020-01-31
CELULA CHINA MED TECH CO LTD
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Problems solved by technology

Differences in 3′-end stability, primer melting temperature (Tm), amplicon length, amplicon GC content, and amplicon flanking region GC content can all contribute to amplification bias
This bias interferes with accurate calculation of copy number for genomic regions of interest and hampers the application of amplicon sequencing to detect small copy number variations

Method used

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  • Method of correcting amplification deviation in amplicon sequencing
  • Method of correcting amplification deviation in amplicon sequencing
  • Method of correcting amplification deviation in amplicon sequencing

Examples

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example 1

[0131] Example 1: Multiplex PCR Amplification Bias Correction for Fetal Aneuploidy Detection

[0132] This article describes a computational method to correct for amplification bias and its application to noninvasive prenatal testing using cell-free maternal DNA to aid in the detection of fetal chromosomal aneuploidy. After correcting for amplification bias in 1855-plex PCR, fetal chromosomal aneuploidy can be detected in maternal blood with a fetal DNA fraction as low as 4%.

[0133] Amplification bias correction for amplicon sequencing was as follows:

[0134] 1. If figure 1 As shown, the coverage of each amplicon is obtained for each sample tested, and then the data is entered into a matrix, with a single row representing a single amplicon and a single column representing a single sample.

[0135] 2. Using the data matrix generated in step 1, generate an amplicon coverage ratio matrix by calculating the coverage ratios for each amplicon combination between the test genomi...

example 2

[0143] Example 2: Multiplex PCR Amplification Bias Correction for Pooled Plasma DNA Samples

[0144] 10 parts of plasma DNA samples were mixed together, and then equally divided into 10 parts for PCR amplification ( Figure 5 ). PCR bias was corrected as described in Example 1, and each data set was processed separately to obtain 10 separate sequencing results. Completing steps 1-4 of Example 1, and then calculating the difference in GC content of the amplicons between each T / R pair (T represents a site in the test region, R represents a site in the reference region), yields Diff amplicon GC, according to the Robust linear regression method to fit the logarithmic normalized amplicon coverage ratio (obtained in step 4 of Example 1) and Diff amplicon GC:

[0145] log(normalized coverage ratio)=β×Diff 扩增子GC +α+ε

[0146] where α is the intercept, β is the slope, and ε is the residual

[0147] As mentioned above, we obtained 10 experimental replicates from the same DNA source...

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Abstract

A method to correct amplification deviation in amplicon sequencing is disclosed. Through steps of amplifying the target nucleic acid to obtain the amplicon coverage of the target nucleic acid, calculating the amplicon coverage ratio between the target nucleic acid in each test genomic region and the target nucleic acid in the reference genomic region, removing outliers, applying a formula to normalize the amplicon coverage ratio, calculating the differences between parameters of the test genomic region amplicon and the reference genomic region amplicon, and applying another formula to fit thedata, and other steps, a regression parameter obtained by fitting is used for correcting the amplification deviation to obtain a normalized amplicon coverage ratio after removing the amplification deviation, thereby eliminating the amplification deviation caused by experimental factors during the multiple PCR amplification process. Elimination of the amplification deviation facilitates accurate calculation of the copy number for a target genomic region, thus enabling detection of minor copy number variation using amplicon sequence data.

Description

technical field [0001] The present invention relates to computational methods for correcting amplification bias in amplicon sequencing. Background technique [0002] Next-generation sequencing, or massively parallel sequencing, typically uses multiplex polymerase chain reaction (PCR)-generated libraries. Differences in 3′-end stability, primer melting temperature (Tm), amplicon length, amplicon GC content, and amplicon flanking region GC content can all contribute to amplification bias. This bias interferes with the precise calculation of copy number for genomic regions of interest and hampers the application of amplicon sequencing to detect small copy number variations. [0003] Bias can be minimized by careful optimization of factors such as primer design, annealing temperature, buffer composition, and number of PCR cycles. See Markoulatos et al. (2002) Journal of Clinical Laboratory Analysis 16:47-51. Alternatively, raw data can be corrected by computational methods th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806G16B25/20
CPCG16B40/10C12Q1/686G16B30/00G16B20/10
Inventor 吴镝张海川
Owner CELULA CHINA MED TECH CO LTD
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