An Amplisenq multigene custom panel was designed to explore all exons of APC (n=16; NM_001128425.1), MLHI (n=17; NM_000249.3), MSH2 (n=16; NM_000251.2), and MSH6 (n=10; NM_000179.2) genes. The details of the target regions as produced by the AmpliSeq designer v2.2.1 are in Additional file 1: Table S1. Thirty nanograms of DNA were used for multiplex PCR amplification,followed by ligation of a specific barcode-sequence to each sample for identification. Emulsion PCR to construct the libraries of clonal sequences was performed with the Ion OneTouch OT2 System (Life Technologies). The quality of the obtained libraries was evaluated by the Agilent 2100 Bioanalyzer on-chip electrophoresis (Agilent Technologies) as previously described [16]. Sequencing of the libraries was performed on personal Genome Machine (PGM, Life Technologies) using the Ion 318 Chip Kit v2. Four samples were processed in each emulsion PCR and sequencing. Data analysis, including alignment to the hg19 human reference genome and veriant calling, was done using the Torrent Suite Software v3.6 (Life Technologies). Filtered veriants were annotated using the SnpEff softwere v3.1. Alignments were visually verified with the Integrative Genomics Viewer (IGV) v.2.2 (Broad Institute). Analysis of blind regions (where automated veriant calling is hindered by sequencing errors due to homopolymers or amplification artifacts) was executed as follows: the COSMIC data-base of SNPs and small INDELs was converted to a Hotspots file and used to guide variant calling. In this way, the variant caller is forced to analyze a given hotspot co-ordinate; if there is no mutation, the software outputs that the position is "reference"; otherwise it outputs the mutation detected. If there are problems in the sequence at that position, the software outputs a "no call" value, explaining why variant calling failed (strand bias, quality of bases,noise in the sequence, low coverage). All the positions where a clear variant/reference status could not be called were further inspected by visual verification of the alignment file to ascertain whether the "no call" tatus was due to artifacts or homopolymer misalignment.