mothur

Problem with pre.cluster or chimera.vsearch

Hi,
I have tried to use the command pre.cluster or chimera.vsearch and each time the Mothur program crash and close the program. I have two different sets of sequences, one for 16S and another for Fungi. And for both types of the sequence appears the same problems.

Here, I pasted my program
Windows version

Using Boost
mothur v.1.44.3
Last updated: 8/27/20
by
Patrick D. Schloss

Department of Microbiology & Immunology

University of Michigan

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 >
make.contigs(processors=24, file=FunBW.files)

Using 24 processors.

[WARNING]: group 1-ITS contains illegal characters in the name. Group names should not include :, -, or / characters. The ‘:’ character is a special character used in trees. Using ‘:’ will result in your tree being unreadable by tree reading software. The ‘-’ character is a special character used by mothur to parse group names. Using the ‘-’ character will prevent you from selecting groups. The ‘/’ character will created unreadable filenames when mothur includes the group in an output filename.

[WARNING]: group 10-ITS contains illegal characters in the name. Group names should not include :, -, or / characters. The ‘:’ character is a special character used in trees. Using ‘:’ will result in your tree being unreadable by tree reading software. The ‘-’ character is a special character used by mothur to parse group names. Using the ‘-’ character will prevent you from selecting groups. The ‘/’ character will created unreadable filenames when mothur includes the group in an output filename.

[WARNING]: group 11-ITS contains illegal characters in the name. Group names should not include :, -, or / characters. The ‘:’ character is a special character used in trees. Using ‘:’ will result in your tree being unreadable by tree reading software. The ‘-’ character is a special character used by mothur to parse group names. Using the ‘-’ character will prevent you from selecting groups. The ‘/’ character will created unreadable filenames when mothur includes the group in an output filename.

[WARNING]: group 12-ITS contains illegal characters in the name. Group names should not include :, -, or / characters. The ‘:’ character is a special character used in trees. Using ‘:’ will result in your tree being unreadable by tree reading software. The ‘-’ character is a special character used by mothur to parse group names. Using the ‘-’ character will prevent you from selecting groups. The ‘/’ character will created unreadable filenames when mothur includes the group in an output filename.

[WARNING]: group 2-ITS contains illegal characters in the name. Group names should not include :, -, or / characters. The ‘:’ character is a special character used in trees. Using ‘:’ will result in your tree being unreadable by tree reading software. The ‘-’ character is a special character used by mothur to parse group names. Using the ‘-’ character will prevent you from selecting groups. The ‘/’ character will created unreadable filenames when mothur includes the group in an output filename.

[WARNING]: group 3-ITS contains illegal characters in the name. Group names should not include :, -, or / characters. The ‘:’ character is a special character used in trees. Using ‘:’ will result in your tree being unreadable by tree reading software. The ‘-’ character is a special character used by mothur to parse group names. Using the ‘-’ character will prevent you from selecting groups. The ‘/’ character will created unreadable filenames when mothur includes the group in an output filename.

[WARNING]: group 4-ITS contains illegal characters in the name. Group names should not include :, -, or / characters. The ‘:’ character is a special character used in trees. Using ‘:’ will result in your tree being unreadable by tree reading software. The ‘-’ character is a special character used by mothur to parse group names. Using the ‘-’ character will prevent you from selecting groups. The ‘/’ character will created unreadable filenames when mothur includes the group in an output filename.

[WARNING]: group 49-ITS contains illegal characters in the name. Group names should not include :, -, or / characters. The ‘:’ character is a special character used in trees. Using ‘:’ will result in your tree being unreadable by tree reading software. The ‘-’ character is a special character used by mothur to parse group names. Using the ‘-’ character will prevent you from selecting groups. The ‘/’ character will created unreadable filenames when mothur includes the group in an output filename.

[WARNING]: group 5-ITS contains illegal characters in the name. Group names should not include :, -, or / characters. The ‘:’ character is a special character used in trees. Using ‘:’ will result in your tree being unreadable by tree reading software. The ‘-’ character is a special character used by mothur to parse group names. Using the ‘-’ character will prevent you from selecting groups. The ‘/’ character will created unreadable filenames when mothur includes the group in an output filename.

[WARNING]: group 50-ITS contains illegal characters in the name. Group names should not include :, -, or / characters. The ‘:’ character is a special character used in trees. Using ‘:’ will result in your tree being unreadable by tree reading software. The ‘-’ character is a special character used by mothur to parse group names. Using the ‘-’ character will prevent you from selecting groups. The ‘/’ character will created unreadable filenames when mothur includes the group in an output filename.

**** Exceeded maximum allowed command warnings, silencing warnings ****

Processing file pair 1-ITS_S49_L001_R1_001.fastq - 1-ITS_S49_L001_R2_001.fastq (files 1 of 24) <<<<<
Making contigs…
Done.

It took 90 secs to assemble 194788 reads.

Processing file pair 10-ITS_S58_L001_R1_001.fastq - 10-ITS_S58_L001_R2_001.fastq (files 2 of 24) <<<<<
Making contigs…
Done.

It took 67 secs to assemble 140475 reads.

Processing file pair 11-ITS_S59_L001_R1_001.fastq - 11-ITS_S59_L001_R2_001.fastq (files 3 of 24) <<<<<
Making contigs…
Done.

It took 93 secs to assemble 200061 reads.

Processing file pair 12-ITS_S60_L001_R1_001.fastq - 12-ITS_S60_L001_R2_001.fastq (files 4 of 24) <<<<<
Making contigs…
Done.

It took 111 secs to assemble 221963 reads.

Processing file pair 2-ITS_S50_L001_R1_001.fastq - 2-ITS_S50_L001_R2_001.fastq (files 5 of 24) <<<<<
Making contigs…
Done.

It took 120 secs to assemble 232707 reads.

Processing file pair 3-ITS_S51_L001_R1_001.fastq - 3-ITS_S51_L001_R2_001.fastq (files 6 of 24) <<<<<
Making contigs…
Done.

It took 127 secs to assemble 235322 reads.

Processing file pair 4-ITS_S52_L001_R1_001.fastq - 4-ITS_S52_L001_R2_001.fastq (files 7 of 24) <<<<<
Making contigs…
Done.

It took 122 secs to assemble 236583 reads.

Processing file pair 49-ITS_S73_L001_R1_001.fastq - 49-ITS_S73_L001_R2_001.fastq (files 8 of 24) <<<<<
Making contigs…
Done.

It took 63 secs to assemble 114754 reads.

Processing file pair 5-ITS_S53_L001_R1_001.fastq - 5-ITS_S53_L001_R2_001.fastq (files 9 of 24) <<<<<
Making contigs…
Done.

It took 70 secs to assemble 155593 reads.

Processing file pair 50-ITS_S74_L001_R1_001.fastq - 50-ITS_S74_L001_R2_001.fastq (files 10 of 24) <<<<<
Making contigs…
Done.

It took 89 secs to assemble 184711 reads.

Processing file pair 51-ITS_S75_L001_R1_001.fastq - 51-ITS_S75_L001_R2_001.fastq (files 11 of 24) <<<<<
Making contigs…
Done.

It took 85 secs to assemble 181312 reads.

Processing file pair 52-ITS_S76_L001_R1_001.fastq - 52-ITS_S76_L001_R2_001.fastq (files 12 of 24) <<<<<
Making contigs…
Done.

It took 74 secs to assemble 163061 reads.

Processing file pair 53-ITS_S77_L001_R1_001.fastq - 53-ITS_S77_L001_R2_001.fastq (files 13 of 24) <<<<<
Making contigs…
Done.

It took 88 secs to assemble 191395 reads.

Processing file pair 54-ITS_S78_L001_R1_001.fastq - 54-ITS_S78_L001_R2_001.fastq (files 14 of 24) <<<<<
Making contigs…
Done.

It took 89 secs to assemble 185533 reads.

Processing file pair 55-ITS_S79_L001_R1_001.fastq - 55-ITS_S79_L001_R2_001.fastq (files 15 of 24) <<<<<
Making contigs…
Done.

It took 75 secs to assemble 132640 reads.

Processing file pair 56-ITS_S80_L001_R1_001.fastq - 56-ITS_S80_L001_R2_001.fastq (files 16 of 24) <<<<<
Making contigs…
Done.

It took 74 secs to assemble 136571 reads.

Processing file pair 57-ITS_S81_L001_R1_001.fastq - 57-ITS_S81_L001_R2_001.fastq (files 17 of 24) <<<<<
Making contigs…
Done.

It took 121 secs to assemble 232386 reads.

Processing file pair 58-ITS_S82_L001_R1_001.fastq - 58-ITS_S82_L001_R2_001.fastq (files 18 of 24) <<<<<
Making contigs…
Done.

It took 93 secs to assemble 187602 reads.

Processing file pair 59-ITS_S83_L001_R1_001.fastq - 59-ITS_S83_L001_R2_001.fastq (files 19 of 24) <<<<<
Making contigs…
Done.

It took 105 secs to assemble 206577 reads.

Processing file pair 6-ITS_S54_L001_R1_001.fastq - 6-ITS_S54_L001_R2_001.fastq (files 20 of 24) <<<<<
Making contigs…
Done.

It took 103 secs to assemble 201671 reads.

Processing file pair 60-ITS_S84_L001_R1_001.fastq - 60-ITS_S84_L001_R2_001.fastq (files 21 of 24) <<<<<
Making contigs…
Done.

It took 93 secs to assemble 182401 reads.

Processing file pair 7-ITS_S55_L001_R1_001.fastq - 7-ITS_S55_L001_R2_001.fastq (files 22 of 24) <<<<<
Making contigs…
Done.

It took 107 secs to assemble 201249 reads.

Processing file pair 8-ITS_S56_L001_R1_001.fastq - 8-ITS_S56_L001_R2_001.fastq (files 23 of 24) <<<<<
Making contigs…
Done.

It took 107 secs to assemble 210000 reads.

Processing file pair 9-ITS_S57_L001_R1_001.fastq - 9-ITS_S57_L001_R2_001.fastq (files 24 of 24) <<<<<
Making contigs…
Done.

It took 105 secs to assemble 206654 reads.

Group count:
1-ITS 194788
10-ITS 140475
11-ITS 200061
12-ITS 221963
2-ITS 232707
3-ITS 235322
4-ITS 236583
49-ITS 114754
5-ITS 155593
50-ITS 184711
51-ITS 181312
52-ITS 163061
53-ITS 191395
54-ITS 185533
55-ITS 132640
56-ITS 136571
57-ITS 232386
58-ITS 187602
59-ITS 206577
6-ITS 201671
60-ITS 182401
7-ITS 201249
8-ITS 210000
9-ITS 206654

Total of all groups is 4536009

It took 2335 secs to process 4536009 sequences.

Output File Names:
FunBW.trim.contigs.fasta
FunBW.scrap.contigs.fasta
FunBW.contigs.report
FunBW.contigs.groups

mothur >
summary.seqs(fasta=E:\Black Walnut project\Fungi BW\FunBW.trim.contigs.fasta)

Using 24 processors.

	Start	End	NBases	Ambigs	Polymer	NumSeqs

Minimum: 1 292 292 0 3 1
2.5%-tile: 1 305 305 0 4 113401
25%-tile: 1 322 322 0 4 1134003
Median: 1 338 338 0 5 2268005
75%-tile: 1 354 354 0 6 3402007
97.5%-tile: 1 580 580 22 11 4422609
Maximum: 1 602 602 118 301 4536009
Mean: 1 354 354 1 5

of Seqs: 4536009

It took 2574 secs to summarize 4536009 sequences.

Output File Names:
E:\Black Walnut project\Fungi BW\FunBW.trim.contigs.summary

mothur >
screen.seqs(fasta=E:\Black Walnut project\Fungi BW\FunBW.trim.contigs.fasta, group=E:\Black Walnut project\Bacteria BW\BacBW.contigs.groups, maxambig=0, maxlength=580)

Using 24 processors.

It took 69 secs to screen 4536009 sequences, removed 904217.

/******************************************/
Running command: remove.seqs(accnos=E:\Black Walnut project\Fungi BW\FunBW.trim.contigs.bad.accnos.temp, group=E:\Black Walnut project\Bacteria BW\BacBW.contigs.groups)
Removed 0 sequences from your group file.

Output File Names:
E:\Black Walnut project\Bacteria BW\BacBW.contigs.pick.groups

/******************************************/

Output File Names:
E:\Black Walnut project\Fungi BW\FunBW.trim.contigs.good.fasta
E:\Black Walnut project\Fungi BW\FunBW.trim.contigs.bad.accnos
E:\Black Walnut project\Fungi BW\BacBW.contigs.good.groups

It took 378 secs to screen 4536009 sequences.

mothur >
summary.seqs(fasta=E:\Black Walnut project\Fungi BW\FunBW.trim.contigs.good.fasta)

Using 24 processors.

	Start	End	NBases	Ambigs	Polymer	NumSeqs

Minimum: 1 300 300 0 3 1
2.5%-tile: 1 305 305 0 4 90795
25%-tile: 1 322 322 0 5 907949
Median: 1 337 337 0 5 1815897
75%-tile: 1 351 351 0 6 2723845
97.5%-tile: 1 520 520 0 11 3540998
Maximum: 1 580 580 0 251 3631792
Mean: 1 345 345 0 5

of Seqs: 3631792

It took 94 secs to summarize 3631792 sequences.

Output File Names:
E:\Black Walnut project\Fungi BW\FunBW.trim.contigs.good.summary

mothur >
unique.seqs(fasta=E:\Black Walnut project\Fungi BW\FunBW.trim.contigs.good.fasta)
3631792 796413

Output File Names:
E:\Black Walnut project\Fungi BW\FunBW.trim.contigs.good.names
E:\Black Walnut project\Fungi BW\FunBW.trim.contigs.good.unique.fasta

mothur >
count.seqs(name=E:\Black Walnut project\Fungi BW\FunBW.trim.contigs.good.names, group=E:\Black Walnut project\Fungi BW\FunBW.contigs.good.groups)

[WARNING]: group 23-ITS contains illegal characters in the name. Group names should not include :, -, or / characters. The ‘:’ character is a special character used in trees. Using ‘:’ will result in your tree being unreadable by tree reading software. The ‘-’ character is a special character used by mothur to parse group names. Using the ‘-’ character will prevent you from selecting groups. The ‘/’ character will created unreadable filenames when mothur includes the group in an output filename.

[WARNING]: group 8-ITS contains illegal characters in the name. Group names should not include :, -, or / characters. The ‘:’ character is a special character used in trees. Using ‘:’ will result in your tree being unreadable by tree reading software. The ‘-’ character is a special character used by mothur to parse group names. Using the ‘-’ character will prevent you from selecting groups. The ‘/’ character will created unreadable filenames when mothur includes the group in an output filename.

[WARNING]: group 60-ITS contains illegal characters in the name. Group names should not include :, -, or / characters. The ‘:’ character is a special character used in trees. Using ‘:’ will result in your tree being unreadable by tree reading software. The ‘-’ character is a special character used by mothur to parse group names. Using the ‘-’ character will prevent you from selecting groups. The ‘/’ character will created unreadable filenames when mothur includes the group in an output filename.

[WARNING]: group 65-ITS contains illegal characters in the name. Group names should not include :, -, or / characters. The ‘:’ character is a special character used in trees. Using ‘:’ will result in your tree being unreadable by tree reading software. The ‘-’ character is a special character used by mothur to parse group names. Using the ‘-’ character will prevent you from selecting groups. The ‘/’ character will created unreadable filenames when mothur includes the group in an output filename.

[WARNING]: group 53-ITS contains illegal characters in the name. Group names should not include :, -, or / characters. The ‘:’ character is a special character used in trees. Using ‘:’ will result in your tree being unreadable by tree reading software. The ‘-’ character is a special character used by mothur to parse group names. Using the ‘-’ character will prevent you from selecting groups. The ‘/’ character will created unreadable filenames when mothur includes the group in an output filename.

[WARNING]: group 5-ITS contains illegal characters in the name. Group names should not include :, -, or / characters. The ‘:’ character is a special character used in trees. Using ‘:’ will result in your tree being unreadable by tree reading software. The ‘-’ character is a special character used by mothur to parse group names. Using the ‘-’ character will prevent you from selecting groups. The ‘/’ character will created unreadable filenames when mothur includes the group in an output filename.

[WARNING]: group 5-ITS contains illegal characters in the name. Group names should not include :, -, or / characters. The ‘:’ character is a special character used in trees. Using ‘:’ will result in your tree being unreadable by tree reading software. The ‘-’ character is a special character used by mothur to parse group names. Using the ‘-’ character will prevent you from selecting groups. The ‘/’ character will created unreadable filenames when mothur includes the group in an output filename.

[WARNING]: group 68-ITS contains illegal characters in the name. Group names should not include :, -, or / characters. The ‘:’ character is a special character used in trees. Using ‘:’ will result in your tree being unreadable by tree reading software. The ‘-’ character is a special character used by mothur to parse group names. Using the ‘-’ character will prevent you from selecting groups. The ‘/’ character will created unreadable filenames when mothur includes the group in an output filename.

[WARNING]: group 23-ITS contains illegal characters in the name. Group names should not include :, -, or / characters. The ‘:’ character is a special character used in trees. Using ‘:’ will result in your tree being unreadable by tree reading software. The ‘-’ character is a special character used by mothur to parse group names. Using the ‘-’ character will prevent you from selecting groups. The ‘/’ character will created unreadable filenames when mothur includes the group in an output filename.

[WARNING]: group 23-ITS contains illegal characters in the name. Group names should not include :, -, or / characters. The ‘:’ character is a special character used in trees. Using ‘:’ will result in your tree being unreadable by tree reading software. The ‘-’ character is a special character used by mothur to parse group names. Using the ‘-’ character will prevent you from selecting groups. The ‘/’ character will created unreadable filenames when mothur includes the group in an output filename.

**** Exceeded maximum allowed command warnings, silencing warnings ****

Removing group: 13-ITS because all sequences have been removed.

Removing group: 14-ITS because all sequences have been removed.

Removing group: 15-ITS because all sequences have been removed.

Removing group: 16-ITS because all sequences have been removed.

Removing group: 17-ITS because all sequences have been removed.

Removing group: 18-ITS because all sequences have been removed.

Removing group: 19-ITS because all sequences have been removed.

Removing group: 20-ITS because all sequences have been removed.

Removing group: 21-ITS because all sequences have been removed.

Removing group: 22-ITS because all sequences have been removed.

Removing group: 23-ITS because all sequences have been removed.

Removing group: 24-ITS because all sequences have been removed.

Removing group: 61-ITS because all sequences have been removed.

Removing group: 62-ITS because all sequences have been removed.

Removing group: 63-ITS because all sequences have been removed.

Removing group: 64-ITS because all sequences have been removed.

Removing group: 65-ITS because all sequences have been removed.

Removing group: 66-ITS because all sequences have been removed.

Removing group: 67-ITS because all sequences have been removed.

Removing group: 68-ITS because all sequences have been removed.

Removing group: 69-ITS because all sequences have been removed.

Removing group: 70-ITS because all sequences have been removed.

Removing group: 71-ITS because all sequences have been removed.

Removing group: 72-ITS because all sequences have been removed.

It took 2691 secs to create a table for 3631792 sequences.

Total number of sequences: 3631792

Output File Names:
E:\Black Walnut project\Fungi BW\FunBW.trim.contigs.good.count_table

mothur >
summary.seqs(fasta=current, count=current)
Using E:\Black Walnut project\Fungi BW\FunBW.trim.contigs.good.count_table as input file for the count parameter.
Using E:\Black Walnut project\Fungi BW\FunBW.trim.contigs.good.unique.fasta as input file for the fasta parameter.

Using 24 processors.

	Start	End	NBases	Ambigs	Polymer	NumSeqs

Minimum: 1 300 300 0 3 1
2.5%-tile: 1 305 305 0 4 90795
25%-tile: 1 322 322 0 5 907949
Median: 1 337 337 0 5 1815897
75%-tile: 1 351 351 0 6 2723845
97.5%-tile: 1 520 520 0 11 3540998
Maximum: 1 580 580 0 251 3631792
Mean: 1 345 345 0 5

of unique seqs: 796413

total # of seqs: 3631792

It took 56 secs to summarize 3631792 sequences.

Output File Names:
E:\Black Walnut project\Fungi BW\FunBW.trim.contigs.good.unique.summary

mothur >
chimera.vsearch(fasta=E:\Black Walnut project\Fungi BW\FunBW.trim.contigs.good.unique.fasta, count=E:\Black Walnut project\Fungi BW\FunBW.trim.contigs.good.count_table)

Using 24 processors.
Checking sequences from E:\Black Walnut project\Fungi BW\FunBW.trim.contigs.good.unique.fasta …

/******************************************/
Running command: split.groups(groups=1-ITS-10-ITS-11-ITS-12-ITS-2-ITS-3-ITS-4-ITS-49-ITS-5-ITS-50-ITS-51-ITS-52-ITS-53-ITS-54-ITS-55-ITS-56-ITS-57-ITS-58-ITS-59-ITS-6-ITS-60-ITS-7-ITS-8-ITS-9-ITS, fasta=E:\Black Walnut project\Fungi BW\FunBW.trim.contigs.good.unique.fasta, count=E:\Black Walnut project\Fungi BW\FunBW.trim.contigs.good.count_table)

Using 24 processors.

Output File Names:

The crashing could be due to mothur running out of memory - that could happen when you run it in windows. Could you monitor the RAM usage when running the command?

Why is it removing groups in count.seqs? Were they removed before?

Thank you for your answer.
I think it is not a PC memory problem, because I tried with only 5 samples and I have the same problem.

Please monitor the ram. I had that problem with four samples I selected for a students module and for those steps it crashed most windows-based computers.

Judging from the output, I suspect you don’t have any samples left by the time you get to pre.cluster or chimera.vsearch. Can you fix your sample names as indicated in the output from count.seqs, re-run and see what you get?

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