Coverage analysis in a targeted amplicon-based next-generation sequencing panel for myeloid neoplasms.

Coverage analysis in a targeted amplicon-based next-generation sequencing panel for myeloid neoplasms.

J Clin Pathol. 2016 Feb 19;

Authors: Yan B, Hu Y, Ng C, Ban KH, Tan TW, Huan PT, Lee PL, Chiu L, Seah E, Ng CH, Koay ES, Chng WJ

Abstract
AIMS: PCR amplicon-based next-generation sequencing (NGS) panels are increasingly used for clinical diagnostic assays. Amplification bias is a well-known limitation of PCR amplicon-based approaches. We sought to characterise lower-performance amplicons in an off-the-shelf NGS panel (TruSight Myeloid Sequencing Panel) for myeloid neoplasms and attempted to patch the low read depth for one of the affected genes, CEBPA.
METHODS: We performed targeted NGS of 158 acute myeloid leukaemia samples and analysed the amplicon read depths across 568 amplicons to identify lower-performance amplicons. We also correlated the amplicon read depths with the template GC content. Finally, we attempted to patch the low read depth for CEBPA using a parallel library preparation (Nextera XT) workflow.
RESULTS: We identified 16 lower-performance amplicons affecting nine genes, including CEBPA. There was a slight negative correlation between the amplicon read depths and template GC content. Addition of the separate CEBPA library generated a minimum read depth per base across the CEBPA gene ranging from 268x to 758x across eight samples.
CONCLUSIONS: The identification of lower-performance amplicons will be informative to laboratories intending to use this panel. We have also demonstrated proof-of-concept that different libraries (TruSight Myeloid and Nextera XT) can be combined and sequenced on the same flow cell to generate additional reads for CEBPA.

PMID: 26896490 [PubMed - as supplied by publisher]

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