CDH1 Mutation Screening through NGS

Somatic mutations were searched in the promoter region (865 bp) and the 16 exons of the CDH1 gene, including splice sites and adjacent intronic regions. The sequencing reaction was performed through next generation sequencing (NGS) with the Roche 454/GS Junior platform (details in the Additional file 1). The reads were aligned to the reference sequence GRCh38-Chr16 using the BWA-MEM algorithm [7 (link)]. All the variants considered were represented in more than 50 reads and had a value > 100 on the Phred quality score.
The variants were corroborated through capillary sequencing with the Abi Prism 310 Genomic Sequencer, using the BigDye™ Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific Inc.©). Bioinformatics analysis used to characterize the identified variants included three tools: PROMO v.3.0.2 [8 (link), 9 (link)], Human Splicing Finder v.3.0 (HSF) [10 (link)] and Translate ExPASy [11 (link)].