2.2 | Primer design
Initial ctenophore COI primers were designed based on COIsequences from our unpublished ctenophore transcriptome data, published mitochondrial sequences of Mnemiopsis leidyi (GenBank accession #: NC016117, JF760210), Pleurobrachia pileus (GenBank accession #: JF760211), and the ctenophores which we could successfully amplify with ‘Folmer primers’ (Folmer et al. 1994) (Figure 1). RNA extractions, transcriptome sequencing, and analyses were conducted as previously described (Francis et al. 2013). We designed primers for the mitochondrial COI gene with Primer3 (Untergasser et al. 2012) within Geneious Prime (v. 2020.2.3, www.geneious.com), using PrimerQuest (www.idtdna.com), or by eye based on nucleotide alignments. Primer design was an iterative process, with multiplexed PCR assays suggesting which variants and combinations of forward and reverse primers worked best. Primer positions were numbered relative to the sites as they occur in M. leidyi (JF760210; Pett et al.2011), as indicated in Figure 1.