Cichlid RNA-seq Analysis Workflow

Raw FASTQ files were processed using cutadapt to 1) trim sequences from the SmartSeq kit, Illumina TruSeq Indexed adapter, and both poly-A and poly-T contamination and 2) exclude sequences shorter than 25bp. The remaining sequence reads were then aligned to the Metriaclima zebra (Malawi cichlid) reference genome UMD2A^{23 (link),52 (link)} using HiSat2^{53 (link)}. Input samples had a slightly higher alignment rates on average than IP samples (85.5% versus 70.5%). Although PC samples had a higher alignment rate than MC samples (85.2% versus 70.7%), this was not driven by sequence divergence between the species and the MZ reference genome, but rather by sample quality. There is no statistically significant difference between the alignment rate between the two species to the M. zebra (reference) genome versus ‘consensus’ genome sequences generated for those species (χ²(2) = 2.09, p = 0.351) (Streelman lab, unpublished data). Transcripts were quantified using StringTie^{54 (link)} and differential expressional analyses were performed using the Bioconductor package DESeq2^{55 (link)}.