Microbiology, Metagenomics and Bioinformatics

Johan Bengtsson-Palme, University of Gothenburg | Wisconsin Institute for Discovery

Browsing Posts tagged 16S

Metaxa2 update

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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.

Metaxa2 is here!

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The new version of MetaxaMetaxa2 – which I first started talking about more than 1.5 years ago, has finally been determined to be so stable that we can officially release it! The release come around the same time as we submitted a paper describing the changes in it, but I will briefly go through the changes here:

  • Metaxa2 now handles extraction and classification of LSU rRNA sequences in addition to SSU rRNA
  • The classification engine has been completely redesigned, and now enables accurate taxonomic classifications down to the genus – or in some cases – species level
  • The classification database has been updated, and is now based on the SILVA 111 release
  • The Metaxa2 Taxonomic Traversal Tool – metaxa2_ttt – has been added to the package, to ease the counting of rRNA sequences in different organism groups (at various taxonomic levels)
  • Metaxa2 adds support for paired-end libraries
  • It is now possible to directly input of sequences in FASTQ-format to Metaxa2
  • The support for libraries with short read lengths (~100 bp) has been vastly improved (and is now assumed to be the case for default settings)
  • Metaxa2 can do quality pre-filtering of reads in FASTQ-format
  • Metaxa2 adds support for the modern BLAST+ package (although the old blastall version is still default)
  • Compatibility with the HMMER 3.1 beta

Metaxa2 brings together a large set of features that we have been gradually incorporating since 2011, many of which have been dependent on each other. Most of the new features and changes are thoroughly explained in the manual. While we hope Metaxa2 is bug free, there will likely be bugs caused by usage scenarios we have not envisioned. I therefore encourage anyone who come across some unexpected behavior to send me an e-mail. Especially, I would like to know about how the software performs using HMMER 3.1 and BLAST+, where testing has been limited compared to older parts of the code.

We hope that you will find Metaxa2 useful, and that it will bring taxonomic assessment of metagenomes another step forward! Metaxa2 can be downloaded here.

As you might be aware, a new version of HMMER is out since late May. You might wonder how Metaxa (relying on HMMER3) will work if you update to the new version of HMMER, and I have finally got around to test it! The answer, according to my somewhat limited testing, is that Metaxa 1.1.2 seems to be working fine with HMMER 3.1.

You might need to go into the database directory (“metaxa_db”; should be located in the same directory as the Metaxa binaries), and remove all the files ending with suffixes .h3f .h3i .h3m and .h3p inside the “HMMs” directory. On most installation, this should not be necessary. Myself, I just plugged HMMER 3.1 in and started Metaxa, but if you get error messages complaining that “Error: bad format, binary auxfiles, .hmm:
binary auxfiles are in an outdated HMMER format (3/b); please hmmpress your HMM file again”, then you should try removing the files and re-running Metaxa. This might especially be a problem on older Metaxa versions. [Update: Note that this fix will likely not work with ITSx!]

Bear in mind that I have not run thorough testing on Metaxa and HMMER 3.1, and probably won’t for the 1.1.2 version, since there’s a 2.0 version waiting just around the corner…

Additionally, if you experience problems with Megraft, you should try the same fix as for Metaxa, but with the Megraft database directory instead. Regarding ITSx, a minor update will be released very soon, which also will address HMMER 3.1b compatibility. [Update: See this post for how to work around HMMER 3.1 problems with ITSx.]

Happy barcoding everyone!

A long time ago, we (Martin Eriksson, Martin Hartmann, Henrik Nilsson and me) were invited to write an overview on Metaxa for the Encyclopedia of Metagenomics. I guess the workload for pulling such a project off is huge, so there’s no surprise that it has taken a while for it to be accepted, but now it is available for consumption.

Meanwhile, Metaxa have been getting regular updates, and I hope to soon be able to show you a new major update to the software, bringing it up to the next generation of metagenomics. More on that soon.

  • Bengtsson-Palme J, Hartmann M, Eriksson KM, Nilsson RH: Metaxa, overview. In:Nelson K. (Ed.) Encyclopedia of Metagenomics: SpringerReference (www.springerreference.com). Springer-Verlag Berlin Heidelberg (2013). [Link]

You might remember that I a long time ago promised a minor update to Megraft. I then forgot about actually posting the update. So it’s very much about time, the updated 1.0.2 version of Megraft. The new thing in this version is improved handling of sequences with N’s (unknown bases) in them, and improved handling of sequences with strange sequence IDs (which sometimes have confused Megraft 1.0.1). The update can be downloaded here.

Some users have asked me to fix a table output bug in Metaxa, and I have finally got around to do so. The fix is released today in the 1.1.2 Metaxa package (download here). This version also brings an updated manual (finally), as the User’s Guide has lagged behind since version 1.0. Please continue to report bugs to metaxa [at sign] microbiology [dot] se

Download the Metaxa package

Read the manual

I just learned from Research in Microbiology that the paper on our software Megraft has now been assigned a volume and an issue. The proper way of referencing Megraft should consequently now be:

Bengtsson J, Hartmann M, Unterseher M, Vaishampayan P, Abarenkov K, Durso L, Bik EM, Garey JR, Eriksson KM, Nilsson RH: Megraft: A software package to graft ribosomal small subunit (16S/18S) fragments onto full-length sequences for accurate species richness and sequencing depth analysis in pyrosequencing-length metagenomes. Research in Microbiology. Volume 163, Issues 6–7 (2012), 407–412, doi: 10.1016/j.resmic.2012.07.001[Paper link]

Megraft is currently at version 1.0.1, but I have a slightly updated version in the pipeline which will be made available later this fall.

Yesterday, our paper on Megraft – a software tool to graft ribosomal small subunit (16S/18S) fragments onto full-length SSU sequences – became available as an accepted online early article in Research in Microbiology. Megraft is built upon the notion that when examining the depth of a community sequencing effort, researchers often use rarefaction analysis of the ribosomal small subunit (SSU/16S/18S) gene in a metagenome. However, the SSU sequences in metagenomic libraries generally are present as fragmentary, non-overlapping entries, which poses a great problem for this analysis. Megraft aims to remedy this problem by grafting the input SSU fragments from the metagenome (obtained by e.g. Metaxa) onto full-length SSU sequences. The software also uses a variability model which accounts for observed and unobserved variability. This way, Megraft enables accurate assessment of species richness and sequencing depth in metagenomic datasets.

The algorithm, efficiency and accuracy of Megraft is thoroughly described in the paper. It should be noted that this is not a panacea for species richness estimates in metagenomics, but it is a huge step forward over existing approaches. Megraft shares some similarities with EMIRGE (Miller et al., 2011), which is a software package for reconstruction of full-length ribosomal genes from paired-end Illumina sequences. Megraft, however, is set apart in that it has a strong focus on rarefaction, and functions also when the number of sequences is small, which is often the case in 454 and Sanger-based metagenomics studies. Thus, EMIRGE and Megraft seek to solve a roughly similar problem, but for different sequencing technologies and sequencing scales.

Megraft is available for download here, and the paper can be read here.

  1. Bengtsson, J., Hartmann, M., Unterseher, M., Vaishampayan, P., Abarenkov, K., Durso, L., Bik, E.M., Garey, J.R., Eriksson, K.M., Nilsson R.H. (2012). Megraft: A software package to graft
  2. Miller, C. S., Baker, B. J., Thomas, B. C., Singer, S. W., & Banfield, J. F. (2011). EMIRGE: reconstruction of full-length ribosomal genes from microbial community short read sequencing data. Genome Biology, 12(5), R44. doi:10.1186/gb-2011-12-5-r44

I proudly announce that today Metaxa has been officially released. Metaxa is a a software tool for automated detection and discrimination among ribosomal small subunit (12S/16S/18S) sequences of archaea, bacteria, eukaryotes, mitochondria, and chloroplasts in metagenomes and environmental sequence datasets. We have been working on Metaxa for quite some time, and it has now been in beta for about two months. However, it seems to be stable enough for public consumption. In addition, the software package is today presented in a talk at the SocBiN conference in Helsinki.

A more thorough post on the rationale behind Metaxa, and how it works will follow when I am not occupied by the SocBiN conference. A paper on Metaxa is to be published in the journal Antonie van Leeuwenhoek. The  software can be downloaded from here.