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

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.

journal_name

J Clin Pathol

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

doi

10.1136/jclinpath-2015-203580

subject

Has Abstract

pub_date

2016-09-01 00:00:00

pages

801-4

issue

9

eissn

0021-9746

issn

1472-4146

pii

jclinpath-2015-203580

journal_volume

69

pub_type

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