We used BFAST (http://bfast.sourceforge.net) (Homer et al., 2009) as our primary alignment algorithm because it has high sensitivity for aligning the reads on the loci containing small insertions and deletions compared to the reference genome (rn4). We used a TopHat-like strategy (Trapnell et al., 2009) to align the sequencing reads that cross splicing junctions using NGSUtils (http://ngsutils.org/) (Breese and Liu, 2013). After aligning the sequence reads to a filtering index including repeats, ribosome RNA, and other sequences that are not of interest, we conducted a sequence alignment for three levels: genome, known junctions (University of California Santa Cruz Genome Browser), and novel junctions (based on the enriched regions identified in the genomic alignment). We restricted our analysis to the uniquely aligned sequences with no more than two mismatches.
