PT  - JOURNAL ARTICLE
AU  - Yan, Benedict
AU  - Hu, Yongli
AU  - Ng, Christopher
AU  - Ban, Kenneth H K
AU  - Tan, Tin Wee
AU  - Huan, Pei Tee
AU  - Lee, Peak-Ling
AU  - Chiu, Lily
AU  - Seah, Elaine
AU  - Ng, Chin Hin
AU  - Koay, Evelyn Siew-Chuan
AU  - Chng, Wee-Joo
TI  - Coverage analysis in a targeted amplicon-based next-generation sequencing panel for myeloid neoplasms
AID  - 10.1136/jclinpath-2015-203580
DP  - 2016 Sep 01
TA  - Journal of Clinical Pathology
PG  - 801--804
VI  - 69
IP  - 9
4099  - http://jcp.bmj.com/content/69/9/801.short
4100  - http://jcp.bmj.com/content/69/9/801.full
SO  - J Clin Pathol2016 Sep 01; 69
AB  - 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.