Sureselect xt human all exon v7

WES libraries were generated using the Agilent Sure Select XT Exome prep kit with 200 ng of input as per protocol (Agilent). The probe used was SureSelect XT Human All Exon V7 (Agilent). Libraries were normalized by Qubit (Invitrogen) and Tapestation (Agilent) and sequenced on a Novaseq 6000(Illumina) to obtain 400x coverage. For analysis, we adopted IMPACT (Integrating Molecular Profiles with Actionable Therapeutics) pipeline that we previously developed(49 (link)). IMPACT links variants detected from WES to actionable therapeutics. Briefly, IMPACT takes sequence data as input and outputs a VCF file containing predicted deleterious mutations. The sequencing reads were mapped to the human hg19 reference exome using the Burrows-Wheeler Aligner. SAMTools and BCFtools (v1.1) were utilized to detect variants from the BAM file and output into a VCF file. In the IMPACT pipeline, we used ANNOVAR (v2014-11-12) to annotate the variants. Synonymous and intronic variants were removed. Variants were further analyzed by deleterious prediction tools such as SIFT and PolyPhen2. We also focused on 49 genes commonly mutated in AML (http://raindancetech.com/thunderbolts-myeloid-panel/) and cancer-related mutations reported in COSMIC (https://cancer.sanger.ac.uk/cosmic) to infer clonal dynamics in paired diagnosis and relapse specimens for VEN+AZA trial patients.

Genomic DNA was extracted from microdissected formalin-fixed paraffin-embedded (FFPE) tissue using a QIAamp DNA FFPE Tissue Kit (Qiagen). At least 250 ng of genomic DNA was used for each sample. DNA quality was assessed on a 4200 TapeStation (Agilent). Genomic DNA libraries were generated using the SureSelectXT Kit (Agilent Technologies), followed by exon enrichment using the SureSelectXT Human All Exon V7 bait set (Agilent Technologies). The resulting exon-enriched libraries were subjected to paired-end, 100-cycle sequencing performed on a HiSeq 4000 (Illumina). Sequencing results were analysed using an institutionally established pipeline for alignment and calling of single nucleotide variants (SNVs) and insertions or deletions (indels), which were annotated and ranked by putative pathogenicity using a workflow named “medal ceremony” and subsequently manually reviewed [18 (link)–20 (link)].