Design of the sequence target used for the computational screen. The bulk of the sequence is the genomic region for BRCA1 (or BRCA2), each of which is more than 80,000 bp in length. For each mutation, we created a sequence with 100 bp of normal sequence flanking the mutation on either side, and concatenated that sequence to the normal region, as shown on the right below the arrows pointing to mutations. This created an artificial index sequence against which all raw sequence reads were aligned. The alignment program, Bowtie, aligned each read to the location of its best match. Reads containing mutations aligned to the mutated portion of the index on the right, while normal reads aligned to the normal BRCA sequence on the left. The small line segments shown below the index illustrate how the reads pile up along the sequence, with gaps in coverage indicating locations where no read matches the index sequence.
Salzberg and Pertea Genome Biology 2010 11:404 doi:10.1186/gb-2010-11-10-404