Hi Artur, thanks for your response. We are pretty certain it's correct, but if you can see a potential problem with the approach described below, please let me know:

[...] the library was sequenced on a MiSeq desktop sequencer. Mitogenome assembly was performed with MITObim version 1.8 (Hahn et al. 2013) using the 16S rRNA sequence of Mastigaodiaptomus nesus (Accession Number: EU582541.1) as the initial bait template. The mitogenome was manually recircularized and re-oriented to the COI gene prior to submission to MITOS for annotation (Bernt et al. 2013).

The 12S and 16S ribosomal RNAs had a length of 546 bp and 527 bp, respectively. The 16S rRNA is unusually short, so its length was verified by means of Sanger sequencing. Custom primers were designed (16S F: 5’-TTG TCC GTC ACT CTC TTT TGT-3’, and 16S-R: 5’-AGG AGA AAA CTC ATA CCC AGG T-3’) that anneal to the trnL1 and the atp8 gene, respectively, which flank the 16S rRNA. The 16S rRNA was amplified via PCR (Bucklin et al. 1995) and sequenced following Jooste et al. (2018), and its short length was confirmed.

Thanks Artur, we'll explore these programs. We just found that a large chunk of the 16S sequence of the sister taxon isn't 16S at all (this is a published mitogenome!), so together with the alternative sofware we should be able to get to the bottom of this!

agree with@Artur Burzynski

regards

Thanks, luck is definitely needed here as we try to find the rest of the 16S. And thanks for keeping us from publishing more nonsense. The system is working!

After some more analysis we are now pretty sure that several previously published 16S sequences contain quite a bit of sequence that is most likely not 16S (attached). The 5' portion aligns well in all of them, but when we move beyond the 3' end of our read, they become unalignable. I'd appreciate your thoughts on this.

Thanks Artur, your input is much appreciated. We had some independent input concerning our short 16S, they suggested that this is definitely stable, and the 16S of reasonably closely related species is similarly short. So we have decided to submit this, but adding a "to the best of our knowledge" comment concerning this issue.

Makes sense, and thanks for your paper. The problem is that even if the other half is present elsewhere in the mitogenome, the fact that the corresponding sequences in the other copepods are so divergent that there is not even the tiniest bit of conserved sequence that we could look for makes this virtually impossible. Published 16S sequences used for popgen or phylogenetic studies are also not helping because everyone has targeted the same portion of 16S (i.e. the one that aligns to our sequence).

That sounds promising, please let us know once it's available. We could then focus specifically on the 16S issue and publish a refinement of the present mitogenome announcement.