Coverage analysis in a targeted amplicon-based next-generation sequencing panel for myeloid neoplasms. Issue 9 (19th February 2016)

• Record Type: Journal Article
• Authors: Yan, Benedict; Hu, Yongli; Ng, Christopher; Ban, Kenneth H K; Tan, Tin Wee; Huan, Pei Tee; Lee, Peak-Ling; Chiu, Lily; Seah, Elaine; Ng, Chin Hin; Koay, Evelyn Siew-Chuan; Chng, Wee-Joo
• Is Part Of: Journal of clinical pathology. Volume 69:Issue 9(2016)
• Page Start: 801
• Page End: 804
• Publication Date: 2016-02-19
• Subjects: GENETICS -- LEUKAEMIA -- MOLECULAR PATHOLOGY; Pathology -- Periodicals; Pathology, Molecular -- Periodicals; 616.0705
• DOI: 10.1136/jclinpath-2015-203580
• Languages: English
• ISSNs: 0021-9746

Abstract

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 .