In search of rare disease-causing variants, we exome sequenced 22 CRC patients who were diagnosed before the age of 40 years.
In addition, in order to exclude the possibility of finding other candidate variants, we have sequenced by Sanger method the majority of these regions.
Notably, the N-terminal peptides from both H3 variants that we sequenced with high confidence started from the amino acid residue 19 of their translation products (Table 5).
All the N-terminal peptides from both H3 variants that we sequenced from six different subjects were starting from the amino acid residue 19 of the corresponding initial translation product.
To search for new AuxRE variants, we collected sequences of all AuxREs experimentally confirmed to date (Additional file 1).
Last, to validate our filtered set of HLOF variants, we Sanger-sequenced any novel variants that we detected in our samples; these included 33 variants which were neither found in the set of HLOF variants of (2) nor (7), and had no validation record in dbSNP.
To confirm the presence of the c.-28A>G and c.-7C>T variants, we performed sequencing of a 1,067-bp 1,067-bpcregionhe MLH1 promoter (c.-960 to c.107 relacrosstheNMLH10249.3) in constitutional DNA from both promoter and identified three heterozygous variants, namely, c.-960>A, c.-28A>G, and c.-7C>to(Supp. Fig. S1A).
To identify sequence variants, we aligned cleaned sequence reads to the assembled transcriptome using Blastn with a threshold of e < 10-5 and requiring 95% identity and alignment of all but the last five bases on each end of the read.
To evaluate Pacific Biosciences data for variant discovery, we sequenced 177 kb in 61 amplicons from regions across human chromosome 20 using both Pacific Biosciences RS and Illumina MiSeq.
In addition to structural variants, we identify novel sequence insertions and novel sequences that are not well aligned to the consensus human genome reference but have high similarity to other human and primate genomes (identity ≥0.95 and align ratio ≥0.95) (Fig. 1f; see Additional file 2, Supplementary Methods for details).
