Targeted DNA-Seq of Human AML treated with TP-0903
Mutations co-occurring with FLT3-ITD before and during TP-0903 treatment were determined by targeted NGS. Libraries were generated using xGen DNA Lib Prep MC UNI kit and target capture was done using xGen AML Cancer Hybridization Panel in conjunction with a three-gene spike-in pool for ZRSR2, AXL, and ASXL2 (Integrated Q16 DNA Technologies). DNA library preparations were performed according to the manufacturers instructions and sequenced on an Illumina NovaSeq (OSUCCC Genomics Shared Resource). Sequenced reads were aligned to the GRCh38 genome build using the Burrows-Wheeler Aligner (BWA). Picard Tools was used to perform UMI-consensus calling on the aligned reads. The Genome Analysis Toolkit (GATK) was used to realign insertions and deletions in the aligned reads and to perform base quality score recalibration for those realigned regions. GATKs MuTect2 was used to perform variant calling. After variant calling, variants were annotated using SnpEff and vcfanno along with the dbsnp, COSMIC, and gnomad variant databases. The Mucor3 algorithm was used as the baseline for integrative mutation assessment. Visual inspection of all variants was carried out using Integrative Genomics Viewer v.2.8 (Broad Institute). A variant allele fraction (VAF) cutoff of 0.01 was set for reporting mutations.