A couple of days ago, a paper I have co-authored describing an ITS sequence dataset for chimera control in fungi went online as an advance online publication in Microbes and Environments. There are several software tools available for chimera detection (e.g. Henrik Nilsson‘s fungal chimera checker (1) and UCHIME (2)), but these generally rely on the presence of a chimera-free reference dataset. Until now, there was no such dataset is for the fungal ITS region, and we in this paper (3) introduce a comprehensive, automatically updated reference dataset for fungal ITS sequences based on the UNITE database (4). This dataset supports chimera detection throughout the fungal kingdom and for full-length ITS sequences as well as partial (ITS1 or ITS2 only) datasets. We estimated the dataset performance on a large set of artificial chimeras to be above 99.5%, and also used the dataset to remove nearly 1,000 chimeric fungal ITS sequences from the UNITE database. The dataset can be downloaded from the UNITE repository. Thereby, it is also possible for users to curate the dataset in the future through the UNITE interactive editing tools.
- Nilsson RH, Abarenkov K, Veldre V, Nylinder S, Wit P de, Brosché S, Alfredsson JF, Ryberg M, Kristiansson E: An open source chimera checker for the fungal ITS region. Molecular Ecology Resources, 10, 1076–1081 (2010).
- Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics, 27, 16, 2194-2200 (2011). doi:10.1093/bioinformatics/btr381
- Nilsson RH, Tedersoo L, Ryberg M, Kristiansson E, Hartmann M, Unterseher M, Porter TM, Bengtsson-Palme J, Walker D, de Sousa F, Gamper HA, Larsson E, Larsson K-H, Kõljalg U, Edgar R, Abarenkov K: A comprehensive, automatically updated fungal ITS sequence dataset for reference-based chimera control in environmental sequencing efforts. Microbes and Environments, Advance Online Publication (2015). doi: 10.1264/jsme2.ME14121
- Kõljalg U, Nilsson RH, Abarenkov K, Tedersoo L, Taylor AFS, Bahram M, Bates ST, Bruns TT, Bengtsson-Palme J, Callaghan TM, Douglas B, Drenkhan T, Eberhardt U, Dueñas M, Grebenc T, Griffith GW, Hartmann M, Kirk PM, Kohout P, Larsson E, Lindahl BD, Lücking R, Martín MP, Matheny PB, Nguyen NH, Niskanen T, Oja J, Peay KG, Peintner U, Peterson M, Põldmaa K, Saag L, Saar I, Schüßler A, Senés C, Smith ME, Suija A, Taylor DE, Telleria MT, Weiß M, Larsson KH: Towards a unified paradigm for sequence-based identification of Fungi. Molecular Ecology, 22, 21, 5271–5277 (2013). doi: 10.1111/mec.12481
A minor bug in the “its1.full_and_partial.fasta” file has been fixed in a minor update to ITSx (1.0.11) released to day. The bug occasionally caused newline characters at the end of a sequence to be skipped and the next entry to begin at the same row. The bug only manifested itself when ITSx was used with the
--partial option and only in the above mentioned FASTA file. If you have been affected by the bug, you should have noticed as the resulting FASTA file would be considered corrupted by most bioinformatics software. The updated version of ITSx can be downloaded here.
An update to Metaxa2 that has long remained in internal testing has been deemed bug-free (as far as we can tell) and has been uploaded to the Metaxa2 web site. The update brings a slightly improved classifier, and is the first release that we declare full stable, although we have found no problems with the previously available version (release candidate 3). This also means that we take a jump directly from version 2.0, release candidate 3 to version 2.0.1 without passing a final 2.0 release. The update is available here.
After a long delay-time in testing ITSx version 1.0.10 has been made public. The new version patches a bug causing the 3′ anchor not being properly written to file when using the “
--anchor hmm” option. If a number was used for the “
--anchor” option, this bug did not apply. Thus, if you have not been using the “
--anchor” option together with “hmm”, you have not been affected in any way by this bug. Nevertheless, I encourage updating in case you would use the “
--anchor hmm” option in the future. The update can be downloaded here. Happy barcoding!
I would like to sincerely apologize for that I have been terrible at responding to support issues pertaining to ITSx, Metaxa, Atosh etc. lately. I am currently on 50% parental leave and at the same time I am wrapping up three first-author papers, organizing a workshop and preparing a talk. Thus, support issues has been lagging a bit behind the last weeks to be able to cope with everything else. I have been ticking off most (all?) of my support questions the last couple of days, but if I have any remaining issues that I have missed to reply to, please re-send them to me!
I will try to improve response times, but it is hard when I am working less than usual (also, note that I (strangely) don’t get paid for supporting software, so I have to do this on my “sparetime”). My aim is to respond within a few days, so if I have not done so, please resend your e-mail with a friendly reminder that you are waiting for my response. Reminding me will very likely put your question up the priority pile.
So, my advice to becoming dads is: Do take paternal leave. Do take a lot of it. Share responsibilities with your partner. Because what you get back is awesome. (And also you get a good reason not to answer support questions in time.) But finally, don’t plan to wrap up the last couple of year’s worth of work and arrange a conference at the same time as you take out paternal leave. That will only make you feel insufficient at all fronts.
Keep the spirit high!
Another paper I have made a contribution to have just recently been published in Molecular Ecology Resources. The paper (1), which is lead-authored by Xin-Cun Wang and Chang Liu at the Institute of Medicinal Plant Development in Beijing, investigates the usability of the ITS1 and ITS2 as separate barcodes across the Eukaryotes. The study is a large scale meta-analysis comparing available high-quality sequence data in as many taxonomic groups at possible from three different aspects: PCR amplification, DNA sequencing efficiency and species discrimination ability. Specifically, we have looked for the presence of DNA barcoding gaps, species discrimination efficiency, sequence length distribution, GC content distribution and primer universality, using bioinformatic approaches. We found that the ITS1 had significantly higher efficiencies than the ITS2 in 17 of 47 families and 20 of 49 investigated genera, which was markedly better than the performance of ITS2. We conclude that, in general, ITS1 represents a better DNA barcode than ITS2 for a majority of eukaryotic taxonomic groups. This of course doesn’t mean that using the ITS2 or the ITS region in its entirety should be dismissed, but our results can serve as a ground for making informed decisions about which region to choose for your amplicon sequencing project. The results complement what have previously been observed for e.g. fungi, where the difference between ITS1 and ITS2 were much less pronounced (2).
- Wang X-C, Liu C, Huang L, Bengtsson-Palme J, Chen H, Zhang J-H, Cai D, Li J-Q: ITS1: A DNA barcode better than ITS2 in eukaryotes? Molecular Ecology Resources. Early view. doi: 10.1111/1755-0998.12325 [Paper link]
- Blaalid R, Kumar S, Nilsson RH, Abarenkov K, Kirk PM, Kauserud H: ITS1 versus ITS2 as DNA metabarcodes for fungi. Molecular Ecology Resources. Volume 13, Issue2, Page 218-224. doi: 10.1111/1755-0998.12065 [Paper link]
I and one of the other developers of ITSx had a discussion a while ago about that using the
--anchor option should output the “anchor sequences” around the ITS regions also for the full-length output file (given that the
--truncate option is activated). I have today changed ITSx to employ this behaviour, updating it to version 1.0.9. The update also improves sensitivity when using the
--anchor HMM option slightly, and can be downloaded here. Happy barcoding!
ITSx has today been updated, bringing it to version 1.0.8. This update adds the “
--only_full” option, which restricts output in the ITS1, 5.8S and ITS2 files to only the files that contain the full region, i.e. that both surrounding domains have been detected. The update also fixes a bug with the
--anchor option, and can be downloaded here. Happy barcoding!
Last week, I was informed by an ITSx user that the software behaved strangely when input files containing extremely long sequence identifiers were used. The bug is not likely to have affected a majority of users, but in any case it is now fixed, and ITSx can now handle sequence identifiers of any length. The new update brings ITSx to version 1.0.7, and it can be downloaded here. Happy barcoding!
A user informed me of unexpected behavior regarding potentially chimeric sequences in ITSx, and indeed it turned out to contain a bug that over-reported potential chimeras. This bug is totally unrelated to the new version released this week, and exists in all prior ITSx versions. I strongly encourage everyone to update to ITSx 1.0.6.
I would also like to underscore that ITSx is not a chimera-checker. It detects when sequences look unusual, but all such cases should be further investigated. If you follow this practice, you will see that in some cases ITSx might have over-reported chimeras, and in some instances it will have been correct in its suspicions (and thereby you would be largely unaffected by this bug).