title: Coverage analysis in a targeted amplicon-based next-generation sequencing panel for myeloid neoplasms.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Laboratory Medicine, Molecular Diagnosis Centre, National University Health System, Singapore, Singapore.
authors.forename: Benedict
authors.initials: B
authors.lastname: Yan
authors.affiliation: Institute for Infocomm and Research, Agency for Science, Technology and Research, Singapore, Singapore.
authors.forename: Yongli
authors.initials: Y
authors.lastname: Hu
authors.affiliation: Department of Laboratory Medicine, Molecular Diagnosis Centre, National University Health System, Singapore, Singapore.
authors.forename: Christopher
authors.initials: C
authors.lastname: Ng
authors.affiliation: Department of Biochemistry, National University of Singapore, Singapore, Singapore Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore, Singapore.
authors.forename: Kenneth H K
authors.initials: KH
authors.lastname: Ban
authors.affiliation: Department of Biochemistry, National University of Singapore, Singapore, Singapore National Supercomputing Centre, Singapore, Singapore.
authors.forename: Tin Wee
authors.initials: TW
authors.lastname: Tan
authors.affiliation: Department of Laboratory Medicine, Molecular Diagnosis Centre, National University Health System, Singapore, Singapore.
authors.forename: Pei Tee
authors.initials: PT
authors.lastname: Huan
authors.affiliation: Department of Laboratory Medicine, Molecular Diagnosis Centre, National University Health System, Singapore, Singapore.
authors.forename: Peak-Ling
authors.initials: PL
authors.lastname: Lee
authors.affiliation: Department of Laboratory Medicine, Molecular Diagnosis Centre, National University Health System, Singapore, Singapore.
authors.forename: Lily
authors.initials: L
authors.lastname: Chiu
authors.affiliation: Department of Haematology-Oncology, National University Cancer Institute, National University Health System, Singapore, Singapore.
authors.forename: Elaine
authors.initials: E
authors.lastname: Seah
authors.affiliation: Department of Haematology-Oncology, National University Cancer Institute, National University Health System, Singapore, Singapore.
authors.forename: Chin Hin
authors.initials: CH
authors.lastname: Ng
authors.affiliation: Department of Laboratory Medicine, Molecular Diagnosis Centre, National University Health System, Singapore, Singapore Department of Pathology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
authors.forename: Evelyn Siew-Chuan
authors.initials: ES
authors.lastname: Koay
authors.affiliation: 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.
authors.forename: Wee-Joo
authors.initials: WJ
authors.lastname: Chng
mesh_terms: D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
keywords: GENETICS; LEUKAEMIA; MOLECULAR PATHOLOGY