mothur

Fasta read error after chimera.vsearch

Hi Folks,

So, I believe some form of this this problem has been address here on the forums before (see here) but I was unable to enact a fix using the recommendations therein. I am hoping someone can perhaps explain it more plainly for me. I am using mothur v1.44.1. I am using vsearch v2.13.3_macos_x86_64.

To explain things from my perspective: I am attempting to process recent MiSeq data using mothur. I am using the MiSeq SOP tutorial as a guideline. I am following the steps precisely as laid out in the SOP and this is the best reference for my inputs to this point.

Even using the files provided by the tutorial, I cannot get past the chimera search, chimera.vsearch(), without error. I believe this is due to a file mismatch, but am not sure. Since I am following a tutorial with sample files I do not suspect poor data to be the culprit.

I have tried a few variations on chimera.vsearch including using fasta+count as the input, as well as fasta+name+group as inputs. The results are the same.

A portion of my log file, including mothur saved files at the time of crash, is posted below (I am not tech-savvy enough to post them as a hyperlink). This screenshot illustrates the fasta read error and subsequent removal of all groups:

Any advice is greatly appreciated! Please let me know if more information/clarification is need! Cheers!

FROM MOTHUR LOGS:

mothur > get.current()

Current RAM usage: 0.327053 Gigabytes. Total Ram: 16 Gigabytes.

Current files saved by mothur:
accnos=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D148.count_table.accnos
fasta=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.fasta
group=/Users/Brad/Desktop/mothur/stability.contigs.good.good.groups
name=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.names
count=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.count_table
processors=4
summary=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.summary

Current default directory saved by mothur: /Users/Brad/Desktop/

Current working directory: /Users/Brad/

Output File Names:
current_files.summary

mothur > chimera.vsearch(fasta=current, name=current, group=current, dereplicate=t)
Using /Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.fasta as input file for the fasta parameter.
Using /Users/Brad/Desktop/mothur/stability.contigs.good.good.groups as input file for the group parameter.
Using /Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.names as input file for the name parameter.

Using 4 processors.
Checking sequences from /Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.fasta …

/******************************************/
Running command: split.groups(groups=F3D0-F3D1-F3D141-F3D142-F3D143-F3D144-F3D145-F3D146-F3D147-F3D148-F3D149-F3D150-F3D2-F3D3-F3D5-F3D6-F3D7-F3D8-F3D9-Mock, fasta=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.fasta, count=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.count_table)

Using 4 processors.
//
Running command: get.seqs(dups=f, accnos=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D6.count_table.accnos, fasta=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.fastaF3D6)
/
/
Running command: get.seqs(dups=f, accnos=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D144.count_table.accnos, fasta=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.fastaF3D144)
//
Running command: get.seqs(dups=f, accnos=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D0.count_table.accnos, fasta=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.fastaF3D0)
/
/
Running command: get.seqs(dups=f, accnos=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D149.count_table.accnos, fasta=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.fastaF3D149)
Selected 504 sequences from your fasta file.

Output File Names:
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fastaF3D144

/******************************************/
Done.
Selected 603 sequences from your fasta file.

Output File Names:
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fastaF3D6

/******************************************/
Done.
Selected 674 sequences from your fasta file.

Output File Names:
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fastaF3D0

/******************************************/
Done.
Selected 994 sequences from your fasta file.

Output File Names:
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fastaF3D149

//
Done.
/
/
Running command: get.seqs(dups=f, accnos=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D7.count_table.accnos, fasta=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.fastaF3D7)
//
Running command: get.seqs(dups=f, accnos=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D145.count_table.accnos, fasta=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.fastaF3D145)
/
/
Running command: get.seqs(dups=f, accnos=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D1.count_table.accnos, fasta=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.fastaF3D1)
/******************************************/
Running command: get.seqs(dups=f, accnos=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D150.count_table.accnos, fasta=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.fastaF3D150)
Selected 404 sequences from your fasta file.

Output File Names:
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fastaF3D7

/******************************************/
Done.
Selected 628 sequences from your fasta file.

Output File Names:
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fastaF3D145

/******************************************/
Done.
Selected 639 sequences from your fasta file.

Output File Names:
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fastaF3D150

/******************************************/
Done.
Selected 551 sequences from your fasta file.

Output File Names:
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fastaF3D1

//
Done.
/
/
Running command: get.seqs(dups=f, accnos=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D8.count_table.accnos, fasta=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.fastaF3D8)
//
Running command: get.seqs(dups=f, accnos=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D146.count_table.accnos, fasta=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.fastaF3D146)
/
/
Running command: get.seqs(dups=f, accnos=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D141.count_table.accnos, fasta=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.fastaF3D141)
/******************************************/
Running command: get.seqs(dups=f, accnos=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D2.count_table.accnos, fasta=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.fastaF3D2)
Selected 491 sequences from your fasta file.

Output File Names:
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fastaF3D8

/******************************************/
Done.
Selected 627 sequences from your fasta file.

Output File Names:
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fastaF3D146

//
Done.
/
/
Running command: get.seqs(dups=f, accnos=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D9.count_table.accnos, fasta=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.fastaF3D9)
Selected 531 sequences from your fasta file.

Output File Names:
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fastaF3D141

/******************************************/
Done.
Selected 1012 sequences from your fasta file.

Output File Names:
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fastaF3D2

//
Done.
/
/
Running command: get.seqs(dups=f, accnos=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D142.count_table.accnos, fasta=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.fastaF3D142)
//
Running command: get.seqs(dups=f, accnos=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D147.count_table.accnos, fasta=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.fastaF3D147)
/
/
Running command: get.seqs(dups=f, accnos=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D3.count_table.accnos, fasta=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.fastaF3D3)
Selected 622 sequences from your fasta file.

Output File Names:
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fastaF3D9

//
Done.
/
/
Running command: get.seqs(dups=f, accnos=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.Mock.count_table.accnos, fasta=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.fastaMock)
Selected 393 sequences from your fasta file.

Output File Names:
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fastaF3D142

/******************************************/
Done.
Selected 483 sequences from your fasta file.

Output File Names:
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fastaF3D3

/******************************************/
Done.
Selected 1160 sequences from your fasta file.

Output File Names:
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fastaF3D147

//
Done.
/
/
Running command: get.seqs(dups=f, accnos=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D143.count_table.accnos, fasta=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.fastaF3D143)
//
Running command: get.seqs(dups=f, accnos=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D5.count_table.accnos, fasta=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.fastaF3D5)
/
/
Running command: get.seqs(dups=f, accnos=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D148.count_table.accnos, fasta=/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.fastaF3D148)
Selected 133 sequences from your fasta file.

Output File Names:
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fastaMock

/******************************************/
Done.
Selected 407 sequences from your fasta file.

Output File Names:
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fastaF3D143

/******************************************/
Done.
Selected 471 sequences from your fasta file.

Output File Names:
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fastaF3D5

/******************************************/
Done.
Selected 901 sequences from your fasta file.

Output File Names:
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fastaF3D148

/******************************************/
Done.

Output File Names:
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D0.count_table
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.F3D0.fasta
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D1.count_table
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.F3D1.fasta
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D141.count_table
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.F3D141.fasta
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D142.count_table
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.F3D142.fasta
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D143.count_table
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.F3D143.fasta
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D144.count_table
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.F3D144.fasta
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D145.count_table
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.F3D145.fasta
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D146.count_table
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.F3D146.fasta
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D147.count_table
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.F3D147.fasta
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D148.count_table
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.F3D148.fasta
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D149.count_table
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.F3D149.fasta
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D150.count_table
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.F3D150.fasta
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D2.count_table
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.F3D2.fasta
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D3.count_table
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.F3D3.fasta
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D5.count_table
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.F3D5.fasta
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D6.count_table
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.F3D6.fasta
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D7.count_table
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.F3D7.fasta
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D8.count_table
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.F3D8.fasta
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.F3D9.count_table
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.F3D9.fasta
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.Mock.count_table
/Users/Brad/Desktop/mothur/stability.trim.contigs.good.unique.good.filter.unique.precluster.Mock.fasta

Thanks for posting such a detailed question. From the screen output you posted it appears that mothur parsed the data into samples, but is unable to run the vsearch program.

/******************************************/
sh: /Users/Brad/Desktop/vsearch: is a directory

Here are a few things to consider:

  1. Upgrade to our current version 1.44.2.

  2. You can tell the chimera.vsearch command where the vsearch program is using the vsearch parameter.

    mothur > chimera.vsearch(fasta=current, name=current, group=current, dereplicate=t, vsearch=nameOfVsearchExecutableIncludingFullPath)

    in my case vsearch is located in my mothur folder:

    mothur > chimera.vsearch(fasta=current, name=current, group=current, dereplicate=t, vsearch=/Users/sarahwestcott/desktop/mothur/vsearch)

  3. Alternatively, you can set a location for all external tools using the set.dir command. (ie, vsearch, uchime, fasterq-dump, prefetch and blast)

    mothur > set.dir(tools=pathToFolderWithMothur’sTools)

    By default the tools are distributed with mothur’s executable versions. You can also download just the tools here, https://github.com/mothur/mothur/releases/download/v1.44.2/Mothur.tools_OSX.zip.

  4. Are you running Catalina on your mac? If so, apple security may be blocking the vsearch exe from opening because it does not include apple developer signing. To work around this, https://www.cultofmac.com/672576/cant-launch-your-apps-on-macos-catalina-heres-the-fix/.

Kindly,
Sarah

Thank you very much for your prompt response Sarah! With some proper directories management I was able to get things working and the issue is resolved! I will be sure to begin each of my runs by explicitly evaluating and setting my input, output, and tools directories to avoid further issues of this nature (doing so smoothed up various aspects of the process in fact).

Cheers,
jbreil

This topic was automatically closed 10 days after the last reply. New replies are no longer allowed.