mothur

chimera.vsearch error

I have been unable to resolve the error message below. mothur v.1.39.5, vsearch vsearch-2.4.3-linux-x86_64, 64-bit Windows 10.
I am trying to adapt the MiSeq SOP to my own test data for benchmarking.

chimera.vsearch(fasta=testdata.trim.contigs.good.unique.good.filter.unique.precluster.fasta, dereplicate=t)
[ERROR]: The reference parameter is a required.

Thanks,
Robert.

If you don’t provide a name or count file, which includes the abundance information, it expects that you are looking for chimeras in reference mode.

Pat

Dear All,

I started to use mothur this week (running version v.1.45.2, on macOS 11.2.3). I’ve been following the MiSeq SOP tutorial with my data. I try to reactivate this post because I’m having issues with the chimera.vsearch command. The following error appears:

mothur > chimera.vsearch(fasta=Run16_20_31_30.good.filter.precluster.fasta, count=Ru16_20_31_30.good.filter.precluster.count_table, dereplicate=t, processors = 8)

[WARNING]: count is not a valid parameter, ignoring.

The valid parameters are: reference, fasta, name, count, group, processors, seed, inputdir, outputdir, abskew, uchimealns, minh, mindiv, xn, dn, mindiffs, vsearch, and dereplicate.

[WARNING]: dereplicate is not a valid parameter, ignoring.

The valid parameters are: reference, fasta, name, count, group, processors, seed, inputdir, outputdir, abskew, uchimealns, minh, mindiv, xn, dn, mindiffs, vsearch, and dereplicate.

[ERROR]: The reference parameter is a required, aborting.

Using 8 processors.

[ERROR]: did not complete chimera.vsearch.

I would appreciate any help and suggestion.

Thank you in advance,

Best,

Víctor

Did you build mothur from the source or are you running our prebuilt version?

Could you post the full logfile?

Dear Sara,

Thank you very much for your prompt reply.
I think is the prebuilt version, I use the file [Mothur.OSX-10.14.zip] from GitHub v1.45.2/Mothur.OSX-10.14.zip.

I’m having the same issue for chimera.uchime.

Please find attached the last log file:

Mac version

Using ReadLine,Boost,HDF5,GSL
mothur v.1.45.2
Last updated: 4/9/21
by
Patrick D. Schloss

Department of Microbiology & Immunology

University of Michigan
http://www.mothur.org

When using, please cite:
Schloss, P.D., et al., Introducing mothur: Open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol, 2009. 75(23):7537-41.

Distributed under the GNU General Public License

Type ‘help()’ for information on the commands that are available

For questions and analysis support, please visit our forum at https://forum.mothur.org

Type ‘quit()’ to exit program

[NOTE]: Setting random seed to 19760620.

Interactive Mode

mothur > set.dir(input = /Users/victorlopez/Desktop/EMED)
Mothur’s directories:
inputDir=/Users/victorlopez/Desktop/EMED/

mothur >
[ERROR]: You are missing (
[ERROR]: Invalid.

mothur > chimera.vsearch(fasta=Run16_20_31_30.good.filter.unique.precluster.fasta, count=Run16_20_31_30.good.filter.unique.precluster.count_table, dereplicate=t)
[WARNING]: count is not a valid parameter, ignoring.
The valid parameters are: reference, fasta, name, count, group, processors, seed, inputdir, outputdir, abskew, uchimealns, minh, mindiv, xn, dn, mindiffs, vsearch, and dereplicate.
[WARNING]: dereplicate is not a valid parameter, ignoring.
The valid parameters are: reference, fasta, name, count, group, processors, seed, inputdir, outputdir, abskew, uchimealns, minh, mindiv, xn, dn, mindiffs, vsearch, and dereplicate.
[ERROR]: /Users/victorlopez/Desktop/EMED/Run16_20_31_30.good.filter.unique.precluster.fasta is blank, aborting.
[ERROR]: You have no current fasta file and the fasta parameter is required.
[ERROR]: The reference parameter is a required, aborting.

Using 4 processors.
[ERROR]: did not complete chimera.vsearch.

mothur >
[ERROR]: You are missing (
[ERROR]: Invalid.

mothur >
[ERROR]: You are missing (
[ERROR]: Invalid.

mothur > chimera.vsearch(fasta=Run16_20_31_30.good.filter.unique.precluster.fasta, count=Run16_20_31_30.good.filter.unique.precluster.count_table, dereplicate=t, processors = 8)
[WARNING]: count is not a valid parameter, ignoring.
The valid parameters are: reference, fasta, name, count, group, processors, seed, inputdir, outputdir, abskew, uchimealns, minh, mindiv, xn, dn, mindiffs, vsearch, and dereplicate.
[WARNING]: dereplicate is not a valid parameter, ignoring.
The valid parameters are: reference, fasta, name, count, group, processors, seed, inputdir, outputdir, abskew, uchimealns, minh, mindiv, xn, dn, mindiffs, vsearch, and dereplicate.
[ERROR]: /Users/victorlopez/Desktop/EMED/Run16_20_31_30.good.filter.unique.precluster.fasta is blank, aborting.
[ERROR]: You have no current fasta file and the fasta parameter is required.
[ERROR]: The reference parameter is a required, aborting.

Using 8 processors.
[ERROR]: did not complete chimera.vsearch.

mothur > chimera.vsearch(fasta=Run16_20_31_30.good.filter.unique.precluster.fasta, count=Run16_20_31_30.good.filter.unique.precluster.count_table, dereplicate=t, processors = 8)
[WARNING]: count is not a valid parameter, ignoring.
The valid parameters are: reference, fasta, name, count, group, processors, seed, inputdir, outputdir, abskew, uchimealns, minh, mindiv, xn, dn, mindiffs, vsearch, and dereplicate.
[WARNING]: dereplicate is not a valid parameter, ignoring.
The valid parameters are: reference, fasta, name, count, group, processors, seed, inputdir, outputdir, abskew, uchimealns, minh, mindiv, xn, dn, mindiffs, vsearch, and dereplicate.
[ERROR]: /Users/victorlopez/Desktop/EMED/Run16_20_31_30.good.filter.unique.precluster.fasta is blank, aborting.
[ERROR]: You have no current fasta file and the fasta parameter is required.
[ERROR]: The reference parameter is a required, aborting.

Using 8 processors.
[ERROR]: did not complete chimera.vsearch.

mothur >
[ERROR]: You are missing (
[ERROR]: Invalid.

mothur > chimera.vsearch(fasta=Run16_20_31_30.good.filter.unique.precluster.fasta, count=Run16_20_31_30.good.filter.unique.precluster.count_table, dereplicate=t, processors = 8)
[WARNING]: count is not a valid parameter, ignoring.
The valid parameters are: reference, fasta, name, count, group, processors, seed, inputdir, outputdir, abskew, uchimealns, minh, mindiv, xn, dn, mindiffs, vsearch, and dereplicate.
[WARNING]: dereplicate is not a valid parameter, ignoring.
The valid parameters are: reference, fasta, name, count, group, processors, seed, inputdir, outputdir, abskew, uchimealns, minh, mindiv, xn, dn, mindiffs, vsearch, and dereplicate.
[ERROR]: /Users/victorlopez/Desktop/EMED/Run16_20_31_30.good.filter.unique.precluster.fasta is blank, aborting.
[ERROR]: You have no current fasta file and the fasta parameter is required.
[ERROR]: The reference parameter is a required, aborting.

Using 8 processors.
[ERROR]: did not complete chimera.vsearch.

mothur > chimera.vsearch(fasta=Run16_20_31_30.good.filter.precluster.fasta, count=Run16_20_31_30.good.filter.precluster.count_table, dereplicate=t, processors = 8)
[WARNING]: count is not a valid parameter, ignoring.
The valid parameters are: reference, fasta, name, count, group, processors, seed, inputdir, outputdir, abskew, uchimealns, minh, mindiv, xn, dn, mindiffs, vsearch, and dereplicate.
[WARNING]: dereplicate is not a valid parameter, ignoring.
The valid parameters are: reference, fasta, name, count, group, processors, seed, inputdir, outputdir, abskew, uchimealns, minh, mindiv, xn, dn, mindiffs, vsearch, and dereplicate.
[ERROR]: The reference parameter is a required, aborting.

Using 8 processors.
[ERROR]: did not complete chimera.vsearch.

mothur > chimera.vsearch(fasta=Run16_20_31_30.good.filter.precluster.fasta, count=Run16_20_31_30.good.filter.precluster.count_table, dereplicate=t, processors = 8)
[WARNING]: count is not a valid parameter, ignoring.
The valid parameters are: reference, fasta, name, count, group, processors, seed, inputdir, outputdir, abskew, uchimealns, minh, mindiv, xn, dn, mindiffs, vsearch, and dereplicate.
[WARNING]: dereplicate is not a valid parameter, ignoring.
The valid parameters are: reference, fasta, name, count, group, processors, seed, inputdir, outputdir, abskew, uchimealns, minh, mindiv, xn, dn, mindiffs, vsearch, and dereplicate.
[ERROR]: The reference parameter is a required, aborting.

Using 8 processors.
[ERROR]: did not complete chimera.vsearch.

mothur > chimera.vsearch(fasta=Run16_20_31_30.good.filter.precluster.fasta, count=Run16_20_31_30.good.filter.precluster.count_table, dereplicate=t, processors = 8)
[WARNING]: count is not a valid parameter, ignoring.
The valid parameters are: reference, fasta, name, count, group, processors, seed, inputdir, outputdir, abskew, uchimealns, minh, mindiv, xn, dn, mindiffs, vsearch, and dereplicate.
[WARNING]: dereplicate is not a valid parameter, ignoring.
The valid parameters are: reference, fasta, name, count, group, processors, seed, inputdir, outputdir, abskew, uchimealns, minh, mindiv, xn, dn, mindiffs, vsearch, and dereplicate.
[ERROR]: The reference parameter is a required, aborting.

Using 8 processors.
[ERROR]: did not complete chimera.vsearch.

mothur >
[ERROR]: You are missing (
[ERROR]: Invalid.

mothur > chimera.uchime(fasta=Run16_20_31_30.good.filter.precluster.fasta, count=Run16_20_31_30.good.filter.precluster.count_table, dereplicate=t, processors = 8)
[WARNING]: count is not a valid parameter, ignoring.
The valid parameters are: reference, fasta, name, count, group, processors, uchime, strand, seed, inputdir, outputdir, abskew, chimealns, minh, mindiv, xn, dn, xa, chunks, minchunk, idsmoothwindow, dereplicate, maxp, skipgaps, skipgaps2, minlen, maxlen, ucl, and queryfract.
[WARNING]: dereplicate is not a valid parameter, ignoring.
The valid parameters are: reference, fasta, name, count, group, processors, uchime, strand, seed, inputdir, outputdir, abskew, chimealns, minh, mindiv, xn, dn, xa, chunks, minchunk, idsmoothwindow, dereplicate, maxp, skipgaps, skipgaps2, minlen, maxlen, ucl, and queryfract.
[ERROR]: The reference parameter is a required, aborting.

Using 8 processors.
[DEBUG]: uchime location using /Users/victorlopez/Documents/mothur/uchime
[ERROR]: did not complete chimera.uchime.

mothur >
[ERROR]: You are missing (
[ERROR]: Invalid.

mothur > chimera.uchime(fasta=Run16_20_31_30.good.filter.precluster.fasta, count=Run16_20_31_30.good.filter.precluster.count_table, dereplicate=t, processors = 8)
[WARNING]: count is not a valid parameter, ignoring.
The valid parameters are: reference, fasta, name, count, group, processors, uchime, strand, seed, inputdir, outputdir, abskew, chimealns, minh, mindiv, xn, dn, xa, chunks, minchunk, idsmoothwindow, dereplicate, maxp, skipgaps, skipgaps2, minlen, maxlen, ucl, and queryfract.
[WARNING]: dereplicate is not a valid parameter, ignoring.
The valid parameters are: reference, fasta, name, count, group, processors, uchime, strand, seed, inputdir, outputdir, abskew, chimealns, minh, mindiv, xn, dn, xa, chunks, minchunk, idsmoothwindow, dereplicate, maxp, skipgaps, skipgaps2, minlen, maxlen, ucl, and queryfract.
[ERROR]: The reference parameter is a required, aborting.

Using 8 processors.
[DEBUG]: uchime location using /Users/victorlopez/Documents/mothur/uchime
[ERROR]: did not complete chimera.uchime.

mothur > chimera.uchime(fasta=Run16_20_31_30.good.filter.precluster.fasta, count=Run16_20_31_30.good.filter.precluster.count_table, dereplicate=t, processors = 8)
[WARNING]: count is not a valid parameter, ignoring.
The valid parameters are: reference, fasta, name, count, group, processors, uchime, strand, seed, inputdir, outputdir, abskew, chimealns, minh, mindiv, xn, dn, xa, chunks, minchunk, idsmoothwindow, dereplicate, maxp, skipgaps, skipgaps2, minlen, maxlen, ucl, and queryfract.
[WARNING]: dereplicate is not a valid parameter, ignoring.
The valid parameters are: reference, fasta, name, count, group, processors, uchime, strand, seed, inputdir, outputdir, abskew, chimealns, minh, mindiv, xn, dn, xa, chunks, minchunk, idsmoothwindow, dereplicate, maxp, skipgaps, skipgaps2, minlen, maxlen, ucl, and queryfract.
[ERROR]: The reference parameter is a required, aborting.

Using 8 processors.
[DEBUG]: uchime location using /Users/victorlopez/Documents/mothur/uchime
[ERROR]: did not complete chimera.uchime.

mothur > chimera.vsearch(fasta=Run16_20_31_30.good.filter.precluster.fasta, count=Run16_20_31_30.good.filter.precluster.count_table, dereplicate=t, processors = 8)
[WARNING]: count is not a valid parameter, ignoring.
The valid parameters are: reference, fasta, name, count, group, processors, seed, inputdir, outputdir, abskew, uchimealns, minh, mindiv, xn, dn, mindiffs, vsearch, and dereplicate.
[WARNING]: dereplicate is not a valid parameter, ignoring.
The valid parameters are: reference, fasta, name, count, group, processors, seed, inputdir, outputdir, abskew, uchimealns, minh, mindiv, xn, dn, mindiffs, vsearch, and dereplicate.
[ERROR]: The reference parameter is a required, aborting.

Using 8 processors.
[ERROR]: did not complete chimera.vsearch.

mothur > quit()




Detected 42 [ERROR] messages, please review.




<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<^>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<^>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<^>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
Detected 22 [WARNING] messages, please review.
<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<^>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<^>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<^>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

Hmm… that’s strange. I am able to run it on my test machines without issue.

Are you copying and pasting the command into the terminal? If so, could there be hidden characters from formatting?

Are you able to run the command from a batch file?

Dear Sara,
Thank you very much for your all your help. I found some errors in my fasta file. I´ll run it again and let you know how it goes.
Best,
Víctor.