Targeted long-read sequencing of the Ewing sarcoma 6p25.1 susceptibility locus: Sample ES_PacBio_056_bam
Genomic DNA from 38 EwS samples (cell lines and PDX) were used to perform targeted amplifications of the region encompassing both the lead SNP rs7742053 and the 6p25.1 GGAA microsatellite locus. Corresponding 5.7kb amplicons were generated after 30 cycles of PCR amplifications using long range PrimerStar GXL DNA polymerase (Takara Bio) and checked by capillary electrophoresis on a Fragment Analyzer instrument (Agilent). 60ng of each amplicon were used to prepare SMRTbell libraries according to “SMRTbell Barcoded Adapter Complete Prep Kit” protocol from Pacific Biosciences. After final quality assessment using BioAnalyzer system with DNA 12000 kit, the pool of libraries was hybridized with PacBio V3 Sequencing primer and bound to the Polymerase from the PacBio V3.0 Binding Kit. The final complex was loaded onto 1 SMRTcell of the Sequel I system. High fidelity long reads (HiFi reads) were generated using Circular Consensus Sequencing tool (CCS), with 3 passes and accuracy of 99% as thresholds from PacBio's open-source SMRT Analysis software (SMRT Link). After demultiplexing using Lima, HiFi reads were aligned to hg19 using minimap2. Phased genotypes based on rs7742053 and GGAA microsatellite locus (chr6:6,837,190-6,837,285) were extracted from each read. GGAA microsatellite structures were determined based on the predominant 24 pattern found for each individual allele