Benedict Yan1, Yongli Hu2, Christopher Ng1, Kenneth H K Ban3, Tin Wee Tan4, Pei Tee Huan1, Peak-Ling Lee1, Lily Chiu1, Elaine Seah5, Chin Hin Ng5, Evelyn Siew-Chuan Koay6, Wee-Joo Chng7. 1. Department of Laboratory Medicine, Molecular Diagnosis Centre, National University Health System, Singapore, Singapore. 2. Institute for Infocomm and Research, Agency for Science, Technology and Research, Singapore, Singapore. 3. Department of Biochemistry, National University of Singapore, Singapore, Singapore Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore, Singapore. 4. Department of Biochemistry, National University of Singapore, Singapore, Singapore National Supercomputing Centre, Singapore, Singapore. 5. Department of Haematology-Oncology, National University Cancer Institute, National University Health System, Singapore, Singapore. 6. 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. 7. 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.
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. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/
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. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/
Authors: Lawrence J Jennings; Maria E Arcila; Christopher Corless; Suzanne Kamel-Reid; Ira M Lubin; John Pfeifer; Robyn L Temple-Smolkin; Karl V Voelkerding; Marina N Nikiforova Journal: J Mol Diagn Date: 2017-03-21 Impact factor: 5.568
Authors: Benedict Yan; Yongli Hu; Kenneth H K Ban; Zenia Tiang; Christopher Ng; Joanne Lee; Wilson Tan; Lily Chiu; Tin Wee Tan; Elaine Seah; Chin Hin Ng; Wee-Joo Chng; Roger Foo Journal: Oncol Lett Date: 2017-02-01 Impact factor: 2.967
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