Benedict Yan1, Yongli Hu2, Christopher Ng1, Kenneth H K Ban3,4, Tin Wee Tan3,5, Pei Tee Huan1, Peak-Ling Lee1, Lily Chiu1, Elaine Seah6, Chin Hin Ng6, Evelyn Siew-Chuan Koay1,7, Wee-Joo Chng6,8,9
1Department of Laboratory Medicine, Molecular Diagnosis Centre, National University Health System, Singapore, Singapore
2Institute for Infocomm and Research, Agency for Science, Technology and Research, Singapore, Singapore
3Department of Biochemistry, National University of Singapore, Singapore, Singapore
4Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore, Singapore
5National Supercomputing Centre, Singapore, Singapore
6Department of Haematology-Oncology, National University Cancer Institute, National University Health System, Singapore, Singapore
7Department of Pathology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
8Cancer Science Institute, National University of Singapore, Singapore, Singapore
9Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
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, includingCEBPA. 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.