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[…] Exome sequencing data was aligned to the human genome (hg19, UCSC assembly, February 2009) using the Burrows–Wheeler–Aligner []. PCR duplicated sequences were removed with Picard-tools [] and BAM files were converted with SAMtools []. Variant calling was done according to GATK Best Practices recommendations [, ] using GATK version 3.1, including local realignment around indels and recalibration of quality scores []. Quality control of called variants was performed using GATK VariantFiltration with parameter settings according to recommendations in SEQanswers exome sequencing analysis guide []. Variants were annotated with ANNOVAR []. Filtering was done using the filtering tool FILTUS version 0.99-9 []. We used two filtering strategies to find causative variant(s). The first approach was based on disease status which would enable us to find variants in potentially novel cancer predisposing genes. The second approach utilized a predefined CRC gene panel which would aid in finding predisposing variants in genes already known to be associated with CRC. The initial filtering steps were identical for the two approaches. These initial steps included removal of all variants that were synonymous, identified in 1000 Genomes Project with MAF <0.001, present in dbSNP build 138 and not flagged as “PASS” after quality control. In the first filtering approach, based on disease status, the remaining variants from 7 individuals (IV:9, IV:10, IV:17, V:4, V:5, V:7 and V:8) classified as “affected” based on their phenotypes were filtered against 1 individual (III:16) classified as “unaffected” (see Online Resource 1 for overview). The remaining individuals (V:2, IV:21, V:10, IV:3 and V:9) were not included in this filtering analysis because they could not be confidently classified as “affected” or “unaffected”. In the second filtering approach, all exome sequenced samples were included and we utilized a predefined panel consisting of genes previously known to be associated with CRC (Online Resource 2). Variants present in the unaffected individual (III:16) were filtered out. For patient V:7 we also applied a panel of genes (Online Resource 3) in which a mutation may predispose to formation of endocrine tumours. Alamut software (Interactive Biosoftware, Rouen, France) was utilized for further annotation of variants. The following tools and measures were used to assess the functional impact at protein level of observed variants: Grantham’s distance [], PhyloP [], SIFT [], MutationTaster [], PolyPhen2 [] and MutationAssessor []. Cutoff values used by the respective prediction programs to determine functional impact of variants is given in Table . Multiple alignment of protein sequences was performed with Clustal Omega [] and ESPript 3.0 []. Domains were annotated according to Shevelev and Hübscher []. Active site residues were annotated according to the Conserved Domains Database (CDD) []. Known variants were annotated according to data from COSMIC v71 [], ExAC Version 0.2 [] and dbSNP Build 142 [].All variants identified in the present study and reported here have been submitted to LOVD 3.0 shared installation ( [...] DNA from EDTA-preserved whole blood or paraffin-embedded tissue was analysed to confirm the variants c.1373A>T (POLE), c.1739T>C (BMPR1A), c.458C>T (EXO1), c.1100del (CHEK2) and c.5265del (LAMB4) detected by exome sequencing, and to test additional family members for the respective variants. PCR was performed using AmpliTaq Gold® 360 MasterMix and 360 GC Enhancer (Life Technologies). Cycle sequencing reaction was performed with BigDye® Terminator v3.1 (Life Technologies) and subsequent capillary electrophoresis was performed by the 3130xl Genetic Analyzer (Life Technologies). Sanger sequencing data was analysed using SeqScape Software v2.5 (Life Technologies). […]

Pipeline specifications

Software tools BWA, Picard, SAMtools, GATK, ANNOVAR, FILTUS, PHAST, SIFT, MutationTaster, PolyPhen, Mutationassessor, Clustal Omega, SeqScape
Databases dbSNP
Applications WES analysis, Sanger sequencing
Diseases Colonic Neoplasms, Neoplasms, Colorectal Neoplasms