Myelofibrosis etiology and transplant outcomes : Sample 10507_PacBio
Targeted PacBio Single Molecule Real-Time (SMRT) sequencing was performed on MF cases to detect JAK2V617F mutations. We followed the SMRT sequencing process protocols as described in the PacBio sequencing manual(https://www.pacb.com/wp-content/uploads/Procedure-Checklist-Preparing-SMRTbell-Libraries-using-PacBio-Barcoded-Universal-Primers-for-Multiplexing-Amplicons.pdf). Briefly, a 2-step PCR, using 10ng of DNA as input, was conducted. In step 1, template-specific primers amplified the region of chr9:5067166-5074380 in GRCh38. Next, in step 2, unique barcode sequences were incorporated onto each sample, using universal tags, for multiplexing. Products were purified and quantified with an additional normalization step to ensure equal concentration of each sample prior to pooling, and hairpin adapters were ligated to the ends of each amplicon pool (up to 384 samples) during the SMRT bell library preparation. Each pooled library had primer annealing and polymerase binding performed according to the protocol and sequenced on 1 Sequel II SMRT Cell 8M. After sequencing, Circular Consensus Sequencing (CCS) read generation was performed with set criteria, including a minimum of 3 passes and accuracy of 99%. CCS reads were used as input to lima (https://github.com/PacificBiosciences/barcoding) to demultiplex the pooled samples, and then aligned to GRCh38 using pbmm (https://github.com/PacificBiosciences/pbmm2).