Coverage analysis in a targeted amplicon-based next-generation sequencing panel for myeloid neoplasms

Abstract

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.

Integration of Technical, Bioinformatic, and Variant Assessment Approaches in the Validation of a Targeted Next-Generation Sequencing Panel for Myeloid Malignancies

“…Other groups have identified alternative approaches to improve sequence coverage at the CEBPA locus, by long-range PCR to capture the entire CEBPA exonic region followed by Nextera (Illumina) library preparation and inclusion for sequencing as part of the TSMP workflow. 38 The TSMP was also not capable of detecting FLT3-ITDs greater than 33 bp in size because of limitations in the amplicon-based technology that restrict the generation/ sequencing of amplicons containing large insertions as well as limitations in bioinformatic tools in aligning and calling larger insertions/deletions. There are efforts to improve upon informatics tools that can be used for detection of insertion/deletions to call indels up to a size limit of 102 bp.…”

Clinical implications ofDNMT3Amutations in a Southeast Asian cohort of acute myeloid leukaemia patients

“…In brief, targeted DNA sequencing was performed using the TruSeq Custom Amplicon assay for the TruSight Myeloid Sequencing Panel (Illumina) as previously described 29 30. The 54 genes assessed via the TruSight Myeloid Sequencing Panel include ABL1, ASXL1, ATRX, BCOR, BCORL1, BRAF, CALR, CBL, CBLB, CBLC, CDKN2A, CEBPA, CSF3R, CUX1, DNMT3A, ETV6, EZH2, FBXW7, FLT3, GATA1,GATA2, GNAS, HRAS, IDH1, IDH2, IKZF1, JAK2, JAK3, KDM6A, KIT, KMT2A, KRAS, MPL, MYD88, NOTCH1, NPM1, NRAS, PDGFRA, PHF6, PTEN, PTPN11, RAD21, RUNX1, SETBP1, SF3B1, SMC1A, SMC3, SRSF2, STAG2, TET2, TP53, U2AF1, WT1 and ZRSR2.…”

CEBPAmutational analysis in acute myeloid leukaemia by a laboratory-developed next-generation sequencing assay

“…The TMSP library preparation was also performed on 122 samples as previously described 5. In brief, quantitation of gDNA was performed using a Qubit fluorometer (Life Technologies, Carlsbad, California, USA) with 50 ng input per sample.…”

“…Due to the high GC content of the gene leading to suboptimal amplification efficiency, we observed poor coverage and read depth in this gene using the TruSight Myeloid Sequencing Panel (TMSP) 5. Hence, we designed a CEBPA -specific NGS protocol, CEBPA- specific Nextera XT amplicon workflow (CEBNX) to overcome these challenges.…”