NGS in FACSorted populations from AML patients

FACS-enriched bone marrow cell populations from 22 AML patients were analyzed using a custom panel targeting 56 genes recurrently mutated in myeloid neoplasms. DNA was released from a median of 60,000 sorted cells by incubation with 2.5 μL proteinase K and 2 μL RNase A (Thermo Fisher Scientific) for 30 minutes at 37°C, purified using 2× AMPure XP beads (Beckman Coulter, Brea, CA), and stored in pH 8 elution buffer at −20°C. DNA quantity was measured using the Qubit dsDNA BR Assay on a Qubit 3.0 fluorometer (Thermo Fisher Scientific), and quality was assessed using the Genomic DNA ScreenTape assay on a 4200 TapeStation system (Agilent Technologies, Santa Clara, CA). Libraries were prepared from at least 20 ng of DNA according to the SOPHiA GENETICS protocol and sequenced on an Illumina MiSeq using 2 × 251-cycle paired-end reads. At least 95% of target bases achieved ≥1,000× coverage. FASTQ files were analyzed in SOPHiA DDM using the GRCh37/hg19 reference genome for alignment, variant calling, and annotation. Variants were retained when the variant allele frequency was ≥2%, coverage was ≥100 reads, and the alternate allele was supported on both strands. Synonymous, intronic, intergenic, 5′ untranslated-region, and recurrent panel-artifact calls were excluded. The remaining variants were classified according to Association for Molecular Pathology and American College of Medical Genetics and Genomics guidance using VarSome, ClinVar, COSMIC, and dbSNP. Only pathogenic or likely pathogenic variants were analyzed. All retained calls were visually confirmed in Integrative Genomics Viewer v2.11.3, and only variants previously reported as somatic in hematopoietic or myeloid neoplasms were retained.

• Type: Cancer Genomics
• Archive: European Genome-phenome Archive (EGA)