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. 2017 Dec 14;18(1):554.
doi: 10.1186/s12859-017-1985-1.

pyAmpli: an amplicon-based variant filter pipeline for targeted resequencing data

Matthias Beyens  1   2 , Nele Boeckx  3   4 , Guy Van Camp  3   4 , Ken Op de Beeck  3   4 , Geert Vandeweyer  3
Affiliations

pyAmpli: an amplicon-based variant filter pipeline for targeted resequencing data

Matthias Beyens et al. BMC Bioinformatics. .

Abstract

Background: Haloplex targeted resequencing is a popular method to analyze both germline and somatic variants in gene panels. However, involved wet-lab procedures may introduce false positives that need to be considered in subsequent data-analysis. No variant filtering rationale addressing amplicon enrichment related systematic errors, in the form of an all-in-one package, exists to our knowledge.

Results: We present pyAmpli, a platform independent parallelized Python package that implements an amplicon-based germline and somatic variant filtering strategy for Haloplex data. pyAmpli can filter variants for systematic errors by user pre-defined criteria. We show that pyAmpli significantly increases specificity, without reducing sensitivity, essential for reporting true positive clinical relevant mutations in gene panel data.

Conclusions: pyAmpli is an easy-to-use software tool which increases the true positive variant call rate in targeted resequencing data. It specifically reduces errors related to PCR-based enrichment of targeted regions.

Keywords: Germline; Next-generation sequencing; Somatic; Targeted resequencing; Variant filtering.

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Conflict of interest statement

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Not applicable.

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Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
Sashimi target enrichment plots. mTOR Exon 54 coverage for two different target enrichment methods is represented by a Sashimi plot: 1) hybridization capture-based technique, showing more bell-shaped enrichment of random fragments (red histogram) and 2) amplicon- or multiplex PCR-based approach, showing vertical enrichment blocks of identical fragments (blue histogram)
Fig. 2
Fig. 2
Vertical read blocks and variant calling bias visualization. Typical vertically enriched read blocks are illustrated in (2a and 2b). Aspecific fragments, not corresponding to predicted amplicons are shown in (2a) and variants restricted to read ends are shown in (2b). Called variants are indicated by a red dashed rectangular. Reads are given in blue and pink colored horizontal bars, indicating read orientation. Theoretical manufacturer designed Haloplex probes are presented in green colored horizontal bars below their corresponding enriched reads
See this image and copyright information in PMC

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