Targeted sequencing of bone-marrow specimen processed from baseline evaluations for MDS
Sample shipments were received and tracked electronically via Emmes' GlobalTrace specimen tracking system. The Invitrogen Qubit 3.0 fluorometer in combination with the dsDNA High Sensitivity Assay Kit was used for DNA assessment of all samples. All samples passed assessment and were able to proceed to the library construction step. Automated (dual indexes) libraries were constructed with 100-250ng of genomic DNA utilizing the KAPA HTP Library Kit (KAPA Biosystems) according to the manufacturer's instructions. Automated libraries were performed on the SciClone NGS (Perkin Elmer) instrument targeting 200-250bp inserts. Samples were pooled in a 1 to 1 ratio. 60 libraries were pooled at an equimolar ratio by mass prior to the hybrid capture targeting a 5mg library pool Library pools are hybridized with the MMPP panel which is a custom xGen Predesigned Gene Capture Pool (IDT) that spans a 96 gene target region. Capture processing was performed following the company protocol. Samples were then passed to the qPCR group for quantitation before sequencing. KAPA qPCR was used to quantify the libraries and determine the appropriate concentration to produce optimal recommended cluster density on a NovaSeq 6000 (PE 150bp) sequencing run. Samples were sequenced on the Illumina NovaSeq 6000 (PE 150bp) instrument using the same dual index recipe targeting greater than 600X mean depth for each sample. All sequencing runs were completed according the manufacturer's recommendations (Illumina Inc - San Diego - CA) Raw sequencing reads were aligned to human genome reference build GRCh38.u2af1_fix.v1.2020_04_01 using BWA-MEM (Version 0.7.15). Duplicate reads were detected and marked using Picard MarkDuplicates (Version 2.4.1), and the read alignments were coordinate sorted using Sambamba (Version 0.6.4). The sequencing quality scores were then corrected for bias via GATK Base Quality Score Recalibration (BaseRecalibrator and PrintReads) (Version 3.6). Final CRAM files were then created using Samtools (v1.3.1).