Fig. 1
Assessment of assay sensitivity, accuracy, and reproducibility. a DNA of FLT3 internal tandem duplication (FLT3-ITD) positive acute myeloid leukemia (AML) cell lines MOLM-14 and PL-21 was each serially diluted in DNA of the FLT3-ITD negative AML cell line HL-60; replicates were diluted independently. The expected ITDs were detected in all samples, down to a variant allele frequency (VAF) of 6.7 × 10−5. Exact numbers and statistics are presented in the supplementary table S1. b Exemplary alignments created by getITD, showing the two types of ITDs detected by our assay: a non-trailing 21 bp ITD (left) and a trailing 198 bp ITD (right). ITD inserts are highlighted in red; the respective wild-type (WT) tandems are highlighted in green. For the trailing ITD, a lighter green mark presumably duplicated WT sequence that was not actually sequenced. Symbols “|” and “.” connect matching and mismatching bases, respectively; gaps indicating insertions and deletions are annotated with “-”. c Comparison of VAF estimates from PCR- and capillary electrophoresis-based fragment analysis (FA) and our next-generation sequencing (NGS)-based assay for 28 FLT3-ITD positive diagnostic AML samples (left) and two independent analyses of 14/28 FLT3-ITD positive diagnostic AML patient samples (right). Allelic ratios (ARs) obtained by FA were converted to VAFs for this comparison as described in the supplement
