We are very happy to announce the release of mothur v.1.30.0. This is a big release with a number of new commands and feature updates. First, we have updated make.contigs to reflect our development and testing of paired end sequencing on a MiSeq using a dual index approach. Use of this command and the overall MiSeq-based workflow has been added to the stable of analysis examples as the Schloss lab’s miseq sop. We have also posted our wetlab protocol there as well. A manuscript describing the SOPs has been submitted to Applied and Environmental Microbiology. This brings us to our second big new command, which we used to write the MiSeq pipeline manuscirpt: write.paper. We’re all overburdened and the climate of publish or perish has convinced us that this new command will help everyone. We’re working on write.dissertation but it’s not quite cleared our stringent QC standards quite yet. A third important new command is primer.design, which will allow you to create a list of candidate primers/probes designed to groups of bacteria within OTUs or phylotypes.
We’ve also added several other useful commands and a number of new feature updates to commands that you will likely find useful. One of note is the ability to use output from summary.seqs, make.contigs, and align.seqs in screen.seqs. This should give more flexibility and quicker execution times. We’ve demonstrated how to do this in the miseq sop. A second option that many will find useful is the ability to set the flow order in trim.flows and shhh.flows to allow 454’s new acyclic flow order (order=B) and Ion Torrent’s acyclic flow order (order=I). Although we have these options available, we are still encouraging people to stick with the original 454 flow order and to stay away from Ion Torrent. Our testing with mock communities has shown that the new technologies are not able to match the performance we see in the 454 SOP with flow order A or using the MiSeq SOP. The next release will hopefully have more guidance on the best course of action for these platforms. Finally, we added a pacbio option to fastq.info which corrects quality scores of 0 to ambiguous base calls. There should be a PacBio SOP posted prior to the next release as well.
This is a big release and there’s a lot going on trying to figure out the best way to generate an analyze data from these new sequencing platforms. If you would like to stay at the bleeding edge of this progress, consider coming to one of Pat’s workshops in Detroit, MI. The next workshops are April 15-17 and May 6-8. Email Pat for more information. Thanks as always for everyone’s feedback and citations. mothur is far and away the most cited bioinformatics program used to analyze 16S rRNA gene sequences. Thank you!
- make.contigs - updated to reflect new MiSeq SOP
- write.paper - given a sff file or collection of fastq files will write a manuscript for a desired journal
- primer.design - will generate candidate PCR primers for OTUs of interest
- get.dists - selects distances from a phylip or column file related to groups or sequences listed in an accnos file.
- remove.dists - removes distances from a phylip or column file related to groups or sequences listed in an accnos file.
- merge.taxsummary - combines tax summary files.
- pre.cluster - added topdown parameter
- various commands - mothur will change ‘:’ characters in sequence names to ‘_’ to avoid problems in downstream analysis with trees.
- added checks to make sure Windows paralellized commands complete their tasks.
- list.otulabels - added list parameter
- get.otus and remove.otus - added list and shared parameters
- count.groups - creates a summary file
- fastq.info and make.fastq - added illumina1.8+ format
- screen.seqs - added contigsreport, alignreport and summary files to allow files to be screened using their inputs. added minoverlap, ostart, oend, mismatches, maxn, minscore, maxinsert, minsim parameters and optimization types of minoverlap, ostart, oend, mismatches, maxn, minscore, maxinsert, minsim.
- pcr.seqs - added pdiffs
- trim.flows and shhh.flows modified to include order=A, order=B, and order=I.
- dist.shared - added column format to output parameter
- consensus.seqs - changed consensus sequence name from seq1, seq2, ... to OTUlabel.
- trim.seqs - added checkorient parameter
- trim.seqs - can now trim paired barcodes and primers.
- get.sharedseqs - added shared file option and changed unique and shared parameter names to uniquegroups and sharedgroups.
- fastq.info - added pacbio parameter
- seq.error - added count parameter
- chimera.slayer, chimera.uchime, chimera.perseus - with count file and dereplicate=t will create a *.pick.count_table file.
- sub.sample - added taxonomy file.
- seq.error - align=f was not degapping the sequences.
- metastats - error with output filename - fixed in 1.29.1.
- venn - segfault when no shared otus - fixed in 1.29.1.
- Windows version removing ‘\’ if full path was given.
- sffinfo - trimmed the entire sequence if clipQualRight=0.
- pcr.seqs - if file was aligned and both forward and reverse primers were given, reverse primer was not trimmed properly. - fixed 1.29.2
- tree.shared - subsampling with eliminated groups. - fixed 1.29.2
- Catchall - [error]: this command doesn’t create a sabund output file.
- amova, homova - https://forum.mothur.org/viewtopic.php?f=1&t=1919
- get.seqs - dups=f seq name mismatches.
- cluster.split - no longer creates concatenated distance matrix for splitmethod=fasta.
- chimera.slayer, chimera.uchime, chimera.perseus - dereplicate=t, remove.seqs(dups=f) was not removing all redundant chimeras.
- sff.multiple - end of file character added to oligos filenames when the input file did not end in blank line, resulting in cannot find file error.
Changes to wiki
- We renamed the “Schloss SOP” to “454 SOP”. You will be automatically re-directed to the 454 page
- We created the miseq_sop page that describes how to use mothur to process MiSeq generated amplicon data.
- fixed bug with “reset to default” menu option.
- removed autofilled file names of removed commands from file input combo boxes.