2.4 Transcriptome sequencing, assembly and annotation
Libraries were sequenced as 150 bp, paired-end (PE) reads using an
Illumina HiSeq4000 sequencing system, performed by Novogene (Hong Kong,
China). Raw data have been deposited in the NCBI Short Read Archive
under the accessions numbers (SRA: SRR10426055-76). Adapter sequences
were trimmed and low quality reads were filtered using a 5 bp sliding
window with a mean quality threshold of 20 and minimum read length of 36
bp using Trimmomatic v0.36 (Bolger, Lohse, & Usadel, 2011). Read
quality (before and after quality-filtering) was assessed using FastQC
v0.11.5 (Andrews, 2010).
Processed reads were assembled de novo with Trinity v.2.5.1
(Grabherr et al., 2011). Separate reference transcriptomes were created
for B. calyciflorus s.s. and B. fernandoi , using all reads
generated for the respective species. To filter possible contamination,
we used a custom perl script which, using the blastn algorithm
(ncbi-blast-2.6.0; Camacho et al., 2015), assigns all contigs either to
a local algae database (Monoraphidium minutum ,Chlamydomonas reinhardtii and, Cryptomonas sp) or to the
respective B. calyciflorus s.s. genome assembly (Kim et al.,
2018). Transcripts were only assigned as of rotifer origin when the top
hit was to the B. calyciflorus s.s. genome and the bit-score gain
over matches to the next species was >100. The same custom
script was further used to remove ribosomal RNA reads by performing a
blastn search to a local database consisting of 18S and 28S sequences ofBrachionus species downloaded from NCBI. We did not utilize theB. calyciflorus s.s. reference genome for the analyses presented
here, as this would introduce a bias, since the B. fernandoireads would not map as well to the reference as those from B.
calyciflorus s.s..