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

J Clin Pathol

Department of Haematology-Oncology, National University Cancer Institute, National University Health System, Singapore, Singapore Cancer Science Institute, National University of Singapore, Singapore, Singapore Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

Published: September 2016

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.

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Source
http://dx.doi.org/10.1136/jclinpath-2015-203580DOI Listing

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