Submicroscopic Mutation Profiling of Myeloid Leukemias

Submicroscopic mutation profiling of 54 genes frequently mutated in myeloid leukemias was done by the Illuminas TruSight Myeloid Gene Panel and sequenced using the MiSeq system and reagent kit v3 (all from Illumina, San Diego, CA, USA) (Additional file 1: Table S4). Amplicon sequencing library was prepared from 50 ng DNA according to the manufacturer’s instructions with the exception of normalization being done manually. 8–16 samples were sequenced each time and the total DNA input on the flow cell was 15 picomolar. Secondary analysis was performed using MiSeqReporter version 2.4.60.8 (Illumina) mapping to the human genome reference hg19. Sequence alignment of selected variants was manually examined with the Integrative Genomics Viewer (IGV) [27 (link)]. Annotation was done by snpSIFT og snpEFF v 4.1. As no matching normal DNA was available variants with >1% minor allele frequency in the 1000 genomes data were presumed to be germline and removed from further interpretations. Synonymous substitutions, intronic variants not in the splice site and variant interpreted as benign or most likely benign are not included. The variant allele frequency (VAF) was calculated for each mutation as number of variant reads divided by total reads. Cut-off for reported variants for VAF was 8% and read depth 100. Only variants interpreted as pathogenic, probably pathogenic and variants of unknown significance are reported. The nomenclature is according to Human Genome Structural Variation consortium.
Fragment analysis of FLT3 exon 14–15 and NPM1 exon 12 were done as described in [28 (link)] and CEBPA mutation analysis as described previously [28 (link)].