The trimmed read sets from both our data and previous sources were used to make a combined assembly using Trinity 2.6.6 (Grabherr et al., 2011). Reads were normalised with a maximum coverage of 50. In the process of assembly checks, we noted a small amount of Parasteatoda tepidariorum contamination of our transcriptome assembly, likely the result of adaptor mis-assignment (P. tepidariorum was sequenced on the same lane as our ovary sample). This has been removed by comparison to the P. tepidariorum genome (Schwager et al., 2017) and transcriptome (Posnien et al., 2014) resources using megablast and removal of hits, which were empirically obvious, with an E value cutoff of 0. The cleaned transcriptome was used for all further analysis. Metrics regarding the resulting assembly were gathered using the TrinityStats.pl script.

Diamond (Buchfink et al., 2015) was used to perform BLASTx searches for initial annotation, against a locally downloaded version of the NCBI nr database. An E value cutoff of 10−6 was applied, and the --max-target-seqs 1 --outfmt six qseqid stitle evalue --more-sensitive settings. BUSCO v1.1b1 (Simão et al., 2015) was used to assess our transcriptomic assembly content, using the metazoan and eukaryote datasets. Expression levels for each sample were quantified using RSEM (Li and Dewey, 2011) and Bowtie2 (Langmead and Salzberg, 2012) as packaged in the Trinity module (align_and_estimate_abundance.pl script,—est_method RSEM—aln_method bowtie2) to compare individual RNAseq samples with the combined assembly.

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