Whole-exome sequencing of localized anal squamous cell carcinoma in human paraffin samples
WES from 46 FFPE samples of ASCC was performed. Purified DNA was quantified by Picogreen and mean size was determined by gel electrophoresis. Genomic DNA was fragmented by mechanical means (Bioruptor) to a mean size of approximately 200 bp. Then, DNA samples were repaired, phosphorylated, A-tailed and ligated to specific adaptors, followed by PCR-mediated labeling with Illumina-specific sequences and sample-specific barcodes (Kapa DNA library generation kit). Exome capture was performed using the VCRome system (capture size of 37 Mb, Roche Nimblegen) under a multiplexing of 8 samples per capture reaction. Capture was strictly carried out following the manufacturers instructions. After capture, libraries were purified, quantified and titrated by Real Time PCR before sequencing. Samples were then sequenced to an approximate coverage of 4.5 Gb per sample in Illumina-NextSeq NS500 (Illumina Inc.) using 150 cycles (2x75) High Output cartridges.