Total RNA was extracted from tumor specimens with RNeasy Mini Kit (Qiagen, Milan, Italy), then cDNA libraries were synthesized from 250 ng total RNA with TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) according to the manufacturer’s recommendations. Sequencing by synthesis was performed on HiScanSQ sequencer (Illumina) at 75 bp in paired-end mode. Whole-transcriptome sequencing yielded a total of 32 Giga Bases and an average of 84 millions of short reads with an average depth of coverage of 45×
WES was performed on DNA isolated from fresh frozen and FFPE tumor tissue and from matched normal peripheral blood or stomach DNA. Whole exome libraries were prepared applying different protocols and using two different sequencing platforms: Nextera Rapid Capture Exome Enrichment (Illumina) was adopted on five out of 19 samples that were sequenced on Illumina HiScanSQ at 2 × 100 bp read length; eight out of 19 libraries were prepared with Nimblegen SeqCap v02 (Roche, Pleasanton, CA, USA), and six out of 19 with Nimblegen SeqCap v03 (Roche, Pleasanton, CA, USA) and were run on HiSeq2000 Illumina platform at 100 bp in single-end. For all the three capturing systems, the exome enrichment was performed according to manufacturer’s protocols.
WES was performed on DNA isolated from fresh frozen and FFPE tumor tissue and from matched normal peripheral blood or stomach DNA. Whole exome libraries were prepared applying different protocols and using two different sequencing platforms: Nextera Rapid Capture Exome Enrichment (Illumina) was adopted on five out of 19 samples that were sequenced on Illumina HiScanSQ at 2 × 100 bp read length; eight out of 19 libraries were prepared with Nimblegen SeqCap v02 (Roche, Pleasanton, CA, USA), and six out of 19 with Nimblegen SeqCap v03 (Roche, Pleasanton, CA, USA) and were run on HiSeq2000 Illumina platform at 100 bp in single-end. For all the three capturing systems, the exome enrichment was performed according to manufacturer’s protocols.