Using Bowtie 2 (Langmead and Salzberg 2012), reads were aligned to the reference genome sequence UMD 3.1 with an average alignment rate of 97.6%, that covered 99.23% of the genome (supplementary table S1, Supplementary Material online).
Sequencing samples have an average depth of 9.75 million reads, with an average transcriptome alignment rate of 49% (Table S1).
Mapping all RNA-seq reads against both transcriptomes (CLC or Velvet/Oases) revealed that the overall mapping success rate, as a measure for assembly quality, was significantly lower for the Velvet/Oases assembly compared to the CLC assembly (see Additional file 1), with an average overall alignment rate of 91.7 % and 73.4 % for the CLC and Velvet/Oases assembly, respectively.
The alignment across all 18 samples produced a mean Overall Read Alignment Rate of 96.4 ± 0.4 % and a mean Concordant Pair Alignment Rate of 88.0 ± 0.2 %, confirming that the read alignment was excellent and that data was of high quality.
Unfortunately, despite these steps, the alignment rate of each aligner was significantly lower than expected, so to offset this, the fastx toolkit was used to filter out low quality reads (Table 1).
The overall alignment rate of reads to the reference sequence was 97.6% with an average read depth of 12.0× (8.77× to 14.77×).
Library sizes were of 32, 46, and 57 million unique reads, respectively, with alignment rate of 92% in each case.
In these tables, we report the very low alignment rate of Bowtie2, which is at most about 13%.
This approach limits consideration to known SNPs, but equalizes the alignment rate of reads containing known variants.
