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
CN110741094AActive Publication Date: 2020-01-31CELULA CHINA MED TECH CO LTD
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
CELULA CHINA MED TECH CO LTD
Publication Date
2020-01-31

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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.
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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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