Tag: Metaxa2

Last week, a preprint describing a study which I have played a small part in was posted on bioRxiv. This paper (1) uses the Metaxa2 database (2) to tease out how much of an effect mitochondrial rRNA sequences have on studies of bacterial diversity in corals. And it turns out that it matters… a lot. Importantly, by supplementing the taxonomic databases with diverse mitochondrial rRNA sequences from the Metaxa2 database, ~97% of unique unclassified sequences could be resolved as mitochondrial, without increasing the level of misannotation in mock communities. Thus the study not only points to a problem, but also to its solution! You can read it all here.

References

1. Sonnet D, Brown T, Bengtsson-Palme J, Padilla-Gamiño J, Zaneveld JR: The Organelle in the Room: Under-annotated Mitochondrial Reads Bias Coral Microbiome Analysis. bioRxiv, 431501 (2021). doi: 10.1101/2021.02.23.431501 [Link]
2. Bengtsson-Palme J, Hartmann M, Eriksson KM, Pal C, Thorell K, Larsson DGJ, Nilsson RH: Metaxa2: Improved identification and taxonomic classification of small and large subunit rRNA in metagenomic data. Molecular Ecology Resources, 15, 6, 1403–1414 (2015). doi: 10.1111/1755-0998.12399 [Paper link]

Yes, Saturdays are somewhat weird days for software updates, but if you’re doing weekend work anyway, why wait to push bug fixes to the community? A very minor bug-fix update to Metaxa2 was released today, bringing the software to version 2.2.3.

Two things have changed in this version, both related to the genome mode. 1) We fixed a file reading bug in the ‘genome’ mode of the software. This bug caused the last sequence in an input FASTA file not to be read unless there was a newline after it. Since the ‘genome’ mode is rarely used by most users, we suspect not a lot of users have been affected by this bug.
2) While we were at it, we changed the behavior of the ‘genome’ mode to mirror that of the ‘auto’ mode, as the strict genome mode dropped sequences shorter than 2500 bp. We considered this behavior counter-intuitive to what most users would want, and has now changed the ‘genome’ mode to behave the same as the ‘auto’ mode and not drop short sequences.

No other changes have been made in this version. The update can be found at the Metaxa2 software page.

Today, we released a minor update to Metaxa2, bringing it to version 2.2.2. The new version includes some bug fixes related to the Metaxa2 Database Repository, as well as a new “–temp” option allowing the user to specify the location for the temporary files. No other changes have been made in this version.

The update can be found at the Metaxa2 software page.

Exactly two years after we released the Metaxa2 database builder, here’s the first update to the software. Unfortunately, it is just a boring bug fix, but the good part is that brings back compatibility with the new version of HMMER (3.3) released in November 2019 (as noted here). It seems like it is mainly the Database builder which has been impacted with by this incompatibility, but we recommend everyone to update.

We have tried to bug check this version as good as we can to make sure we did not break any features while introducing this new compatibility. We think that this version is bug free, but as we wanted to push this out quickly, please be more observant than usual to odd behaviour, and make sure to report any bugs!

The update can be downloaded here: https://microbiology.se/sw/Metaxa2_2.2.1.tar.gz

Update: There is now an updated version of Metaxa2 that addresses this problem. Find it here.

We have recently discovered that the new version of HMMER (3.3) released in November 2019 have introduced new restrictions that make it partially incompatible with Metaxa2. The most apparent problem is in the Database Builder software, which will not build profiles properly in most cases. Instead, HMMER will return an error and only some profiles will be created.

We do currently not know if this also affects the functionality of Metaxa2 itself. We are currently investigating this.

For now, the solution to this problem is to use the previous version of HMMER (version 3.2.1) while we investigate further. That version can be downloaded here: http://hmmer.org/download.html

I am sorry about not discovering this earlier, this only came to our attention this week!

A few days ago, my attention was turned to a duplicate in the COI database bundled with Metaxa2 2.2. While this duplicate sequence should not cause any troubles for Metaxa2 itself, it has created issues for people using the database itself together with, e.g., QIIME. Therefore, I have today issued a very very minor update to the Metaxa2 2.2 package as well as the entry in the Metaxa2 Database Repository, removing the duplicate sequence. I deemed that this was not significant enough to issue a new version, particularly as no code was changed and it did not cause issues for the software itself, so the version will stay at 2.2 for the time being. Happy barcoding!

Last week, I uploaded a new database to the Metaxa2 Database Repository, called DAIRYdb. DAIRYdb (1) is a manually curated reference database for 16S rRNA amplicon sequences from dairy products. Significant efforts have been put into improving annotation algorithms, such as Metaxa2 (2), while less attention has been put into curation of reliable and consistent databases (3). Previous studies have shown that databases restricted to the studied environment improve unambiguous taxonomy annotation to the species level, thanks to consistent taxonomy, lack of blanks and reduced competition between different reference taxonomies (4-5). The usage of DAIRYdb in combination with different classification tools allows taxonomy annotation accuracy of over 90% at species level for microbiome samples from dairy products, where species identification is mandatory due to the affiliation to few closely related genera of most dominant lactic acid bacteria.

The database can be added to your Metaxa2 (version 2.2 or later) installation by using the following command:

metaxa2_install_database -g SSU_DAIRYdb_v1.1.2

Further adaptations of the DAIRYdb can be found on GitHub and the preprint has been deposited in BioRxiv (1). DAIRYdb was developed by Marco Meola, Etienne Rifa and their collaborators, who also provided most of the text for this post. Thanks Marco for this excellent addition to the database collection!

References

1. Meola M, Rifa E, Shani N, Delbes C, Berthoud H, Chassard C: DAIRYdb: A manually curated gold standard reference database for improved taxonomy annotation of 16S rRNA gene sequences from dairy products. bioRxiv, 386151 (2018). doi: 10.1101/386151
2. Bengtsson-Palme J, Hartmann M, Eriksson KM, Pal C, Thorell K, Larsson DGJ, Nilsson RH: Metaxa2: Improved identification and taxonomic classification of small and large subunit rRNA in metagenomic data. Molecular Ecology Resources, 15, 6, 1403–1414 (2015). doi: 10.1111/1755-0998.12399
3. Edgar RC: Accuracy of taxonomy prediction for 16S rRNA and fungal ITS sequences. PeerJ, 6, e4652 (2018). doi: 10.7717/peerj.4652
4. Ritari J, Salojärvi J, Last L, de Vos WM: Improved taxonomic assignment of human intestinal 16S rRNA sequences by a dedicated reference database. BMC Genomics, 16, 1, 1056 (2015). doi: 10.1186/s12864-015-2265-y
5. Newton ILG, Roeselers G: The effect of training set on the classification of honey bee gut microbiota using the naïve bayesian classifier. BMC Microbiology, 12, 1, 221 (2012). doi: 10.1186/1471-2180-12-221

A few days ago I posted about that Bioinformatics had published our paper on the Metaxa2 Database Builder (1). Today, I am happy to report that PeerJ has published the first paper in which the database builder is used to create a new Metaxa2 (2) database! My colleagues at Ohio State University has used the software to build a database for the COI gene (3), which is commonly used in arthropod barcoding. The used region was extracted from COI sequences from arthropod whole mitochondrion genomes, and employed to create a database containing sequences from all major arthropod clades, including all insect orders, all arthropod classes and the Onychophora, Tardigrada and Mollusca outgroups.

Similar to what we did in our evaluation of taxonomic classifiers used on non-rRNA barcoding regions (4), we performed a cross-validation analysis to characterize the relationship between the Metaxa2 reliability score, an estimate of classification confidence, and classification error probability. We used this analysis to select a reliability score threshold which minimized error. We then estimated classification sensitivity, false discovery rate and overclassification, the propensity to classify sequences from taxa not represented in the reference database.

Since the database builder was still in its early inception stages when we started doing this work, the software itself saw several improvements because of this project. We believe that our work on the COI database, as well as on the recently released database builder software, will help researchers in designing and evaluating classification databases for metabarcoding on arthropods and beyond. The database is included in the new Metaxa2 2.2 release, and is also downloadable from the Metaxa2 Database Repository (1). The open access paper can be found here.

References

1. Bengtsson-Palme J, Richardson RT, Meola M, Wurzbacher C, Tremblay ED, Thorell K, Kanger K, Eriksson KM, Bilodeau GJ, Johnson RM, Hartmann M, Nilsson RH: Metaxa2 Database Builder: Enabling taxonomic identification from metagenomic and metabarcoding data using any genetic marker. Bioinformatics, advance article (2018). doi: 10.1093/bioinformatics/bty482
2. Bengtsson-Palme J, Hartmann M, Eriksson KM, Pal C, Thorell K, Larsson DGJ, Nilsson RH: Metaxa2: Improved identification and taxonomic classification of small and large subunit rRNA in metagenomic data. Molecular Ecology Resources, 15, 6, 1403–1414 (2015). doi: 10.1111/1755-0998.12399
3. Richardson RT, Bengtsson-Palme J, Gardiner MM, Johnson RM: A reference cytochrome c oxidase subunit I database curated for hierarchical classification of arthropod metabarcoding data. PeerJ, 6, e5126 (2018). doi: 10.7717/peerj.5126
4. Richardson RT, Bengtsson-Palme J, Johnson RM: Evaluating and Optimizing the Performance of Software Commonly Used for the Taxonomic Classification of DNA Sequence Data. Molecular Ecology Resources, 17, 4, 760–769 (2017). doi: 10.1111/1755-0998.12628

One of the questions I have received regarding Metaxa2 is if it is possible to use it on other DNA barcodes. My answer has been “technically, yes, but it is a very cumbersome process of creating a custom database for every additional barcode”. Not anymore, the newly introduced Metaxa2 Database Builder makes this process automatic, with the user just supplying a FASTA file of sequences from the region in question and a file containing the taxonomy information for the sequences (in GenBank, NSD XML, Metaxa2 or SILVA-style formats). The preprint (1) has been out for some time, but today Bioinformatics published the paper describing the software (2).

The paper not only details how the database builder works, but also shows that it is working on a number of different barcoding regions, albeit with different results in terms of accuracy. Still, even with seemingly high misclassification rates for some DNA barcodes, the software performs better than a simple BLAST-based taxonomic assignment (76.5% vs. 41.4% correct classifications for matK, and 76.2% vs. 45.1% for tnrL). The database builder has already found use in building a COI database for anthropods (3), and we envision a range of uses in the near future.

As the paper is now published, I have also moved the Metaxa2 software (4) from beta-status to a full-worthy version 2.2 update. Hopefully, this release should be bug free, but my experience is that when the community gets their hands of the software they tend to discover things our team has missed. I would like to thank the entire team working on this, particularly Rodney Richardson (who initiated this entire thing) and Henrik Nilsson. The software can be downloaded here. Happy barcoding!

References

1. Bengtsson-Palme J, Richardson RT, Meola M, Wurzbacher C, Tremblay ED, Thorell K, Kanger K, Eriksson KM, Bilodeau GJ, Johnson RM, Hartmann M, Nilsson RH: Taxonomic identification from metagenomic or metabarcoding data using any genetic marker. bioRxiv 253377 (2018). doi: 10.1101/253377 [Link]
2. Bengtsson-Palme J, Richardson RT, Meola M, Wurzbacher C, Tremblay ED, Thorell K, Kanger K, Eriksson KM, Bilodeau GJ, Johnson RM, Hartmann M, Nilsson RH: Metaxa2 Database Builder: Enabling taxonomic identification from metagenomic and metabarcoding data using any genetic marker. Bioinformatics, advance article (2018). doi: 10.1093/bioinformatics/bty482 [Paper link]
3. Richardson RT, Bengtsson-Palme J, Gardiner MM, Johnson RM: A reference cytochrome c oxidase subunit I database curated for hierarchical classification of arthropod metabarcoding data. PeerJ Preprints, 6, e26662v1 (2018). doi: 10.7287/peerj.preprints.26662v1 [Link]
4. Bengtsson-Palme J, Hartmann M, Eriksson KM, Pal C, Thorell K, Larsson DGJ, Nilsson RH: Metaxa2: Improved identification and taxonomic classification of small and large subunit rRNA in metagenomic data. Molecular Ecology Resources, 15, 6, 1403–1414 (2015). doi: 10.1111/1755-0998.12399 [Paper link]

Due to an extremely embarrassing for-loop error in the classifier of the most recent Metaxa2 beta (beta 8), which was released a few weeks ago, the classifier often would (on certain platforms and configurations) enter an endless loop and hang. I apologize for this mistake, which has been corrected in the new beta 9 released today, available from this download link. No other changes have been made since the previous version. Thanks for your patience (and thanks Kaisa Thorell for first bringing my attention the error!)