# classify.seqs

**URL:** <https://forum.mothur.org/t/classify-seqs/1932>\
**Category:** mothur bugs\
**Created:** [July 22, 2014, 11:22am UTC](https://forum.mothur.org/t/classify-seqs/1932 "2014-07-22T11:22:20Z")\
**Posts on this page:** 4\
**Page:** 1

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**Author:** ![jcauguet](https://avatars.discourse-cdn.com/v4/letter/j/b3f665/32.png) [@jcauguet](https://forum.mothur.org/u/jcauguet)\
**Post date:** [July 22, 2014, 11:22am UTC](https://forum.mothur.org/t/classify-seqs/1932/1 "2014-07-22T11:22:20Z")

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Hi,  
I tried to classify mi Miseq archaea sequences with the greengenes databases (the new one and the old version) with the following command line:

_classify.seqs(fasta=arch.trim.contigs.good.unique.good.filter.unique.precluster.pick.abund.fasta, count=arch.trim.contigs.good.unique.good.filter.unique.precluster.uchime.pick.abund.count\_table, reference=gg\_99.pds.ng.fasta, taxonomy=gg\_99.pds.tax, cutoff=80, processors=12)_

But I get every time the following error message from my server:

\_ModuleCmd\_Load.c(204):ERROR:105: Unable to locate a modulefile for ‘mothur/1.32.1’  
Your job has been killed.  
This may happen if one of the followings hold :

- you exceeded one of the queue/job limits (run time, memory, etc)
- you (or admin) killed the job using qdel
- something bad happened.  
Now, just in case something bad happened, here are the debug information about your job :  
total 4  
-rw-r–r-- 1 jauguet UMR5254 50 22 juil. 13:11 job\_killed  
JOB INFO :  
total 40  
-rw-r–r-- 1 sgeadmin sgeadmin 34 22 juil. 13:05 pe\_hostfile  
-rw-r–r-- 1 sgeadmin sgeadmin 6440 22 juil. 13:05 environment  
-rw-r–r-- 1 sgeadmin sgeadmin 2051 22 juil. 13:05 config  
-rw-r–r-- 1 sgeadmin sgeadmin 6 22 juil. 13:05 pid  
-rw-r–r-- 1 sgeadmin sgeadmin 6 22 juil. 13:05 job\_pid  
-rw-r–r-- 1 sgeadmin sgeadmin 6 22 juil. 13:05 addgrpid  
-rw-r–r-- 1 jauguet UMR5254 0 22 juil. 13:05 error  
-rw-r–r-- 1 jauguet UMR5254 0 22 juil. 13:05 exit\_status  
-rw-r–r-- 1 sgeadmin sgeadmin 319 22 juil. 13:11 usage  
-rw-r–r-- 1 jauguet UMR5254 6556 22 juil. 13:11 trace  
STATUS :  
TRACE :  
07/22/2014 13:05:47 [500:43604]: shepherd called with uid = 0, euid = 500  
07/22/2014 13:05:47 [500:43604]: starting up 2011.11  
07/22/2014 13:05:47 [500:43604]: setpgid(43604, 43604) returned 0  
07/22/2014 13:05:47 [500:43604]: do\_core\_binding: “binding” parameter not found in config file  
07/22/2014 13:05:47 [500:43604]: no prolog script to start  
07/22/2014 13:05:47 [500:43604]: parent: forked “job” with pid 43606  
07/22/2014 13:05:47 [500:43604]: parent: job-pid: 43606  
07/22/2014 13:05:47 [500:43606]: child: starting son(job, /var/spool/sge/ceri025/job\_scripts/6709200, 0);  
07/22/2014 13:05:47 [500:43606]: pid=43606 pgrp=43606 sid=43606 old pgrp=43604 getlogin()=  
07/22/2014 13:05:47 [500:43606]: reading passwd information for user ‘jauguet’  
07/22/2014 13:05:47 [500:43606]: setosjobid: uid = 0, euid = 500  
07/22/2014 13:05:47 [500:43606]: setting limits  
07/22/2014 13:05:47 [500:43606]: RLIMIT\_CPU setting: (soft 18446744073709551615(INFINITY), hard 18446744073709551615(INFINITY)) resulting: (soft 18446744073709551615(INFINITY), hard 18446744073709551615(INFINITY))  
07/22/2014 13:05:47 [500:43606]: RLIMIT\_FSIZE setting: (soft 18446744073709551615(INFINITY), hard 18446744073709551615(INFINITY)) resulting: (soft 18446744073709551615(INFINITY), hard 18446744073709551615(INFINITY))  
07/22/2014 13:05:47 [500:43606]: RLIMIT\_DATA setting: (soft 18446744073709551615(INFINITY), hard 18446744073709551615(INFINITY)) resulting: (soft 18446744073709551615(INFINITY), hard 18446744073709551615(INFINITY))  
07/22/2014 13:05:47 [500:43606]: RLIMIT\_STACK setting: (soft 268435456, hard 268435456) resulting: (soft 268435456, hard 268435456)  
07/22/2014 13:05:47 [500:43606]: RLIMIT\_CORE setting: (soft 0, hard 0) resulting: (soft 0, hard 0)  
07/22/2014 13:05:47 [500:43606]: RLIMIT\_MEMLOCK setting: (soft 18446744073709551615(INFINITY), hard 18446744073709551615(INFINITY)) resulting: (soft 18446744073709551615(INFINITY), hard 18446744073709551615(INFINITY))  
07/22/2014 13:05:47 [500:43606]: RLIMIT\_VMEM/RLIMIT\_AS setting: (soft 8589934592, hard 8589934592) resulting: (soft 8589934592, hard 8589934592)  
07/22/2014 13:05:47 [500:43606]: RLIMIT\_RSS setting: (soft 18446744073709551615(INFINITY), hard 18446744073709551615(INFINITY)) resulting: (soft 18446744073709551615(INFINITY), hard 18446744073709551615(INFINITY))  
07/22/2014 13:05:47 [500:43606]: setting environment  
07/22/2014 13:05:47 [500:43606]: Initializing error file  
07/22/2014 13:05:47 [500:43606]: switching to intermediate/target user  
07/22/2014 13:05:47 [13532:43606]: closing all filedescriptors  
07/22/2014 13:05:47 [13532:43606]: further messages are in “error” and “trace”  
07/22/2014 13:05:47 [13532:43606]: now running with uid=13532, euid=13532  
07/22/2014 13:05:47 [13532:43606]: execvp(/var/spool/sge/ceri025/job\_scripts/6709200, “/var/spool/sge/ceri025/job\_scripts/6709200”)  
07/22/2014 13:11:04 [500:43604]: wait3 returned -1  
07/22/2014 13:11:04 [500:43604]: forward\_signal\_to\_job(): mapping signal 20 TSTP  
07/22/2014 13:11:04 [500:43604]: mapped signal TSTP to signal KILL  
07/22/2014 13:11:04 [500:43604]: queued signal KILL  
07/22/2014 13:11:04 [500:43604]: /SGE/GE2011.11/inra/tools/terminate\_method.sh $job\_owner $job\_pid -\> overriddes kill(-43606, KILL)  
07/22/2014 13:11:04 [500:52460]: starting terminate\_method command: /SGE/GE2011.11/inra/tools/terminate\_method.sh jauguet 43606  
07/22/2014 13:11:04 [13532:52460]: start\_as\_command: pre\_args\_ptr[0] = argv0; “/SGE/GE2011.11/inra/tools/terminate\_method.sh jauguet 43606” shell\_path = “/bin/bash”  
07/22/2014 13:11:04 [13532:52460]: execvp(/bin/bash, “/SGE/GE2011.11/inra/tools/terminate\_method.sh jauguet 43606” “-c” “/SGE/GE2011.11/inra/tools/terminate\_method.sh jauguet 43606”)  
07/22/2014 13:11:05 [500:43604]: wait3 returned -1  
07/22/2014 13:11:05 [500:43604]: forward\_signal\_to\_job(): mapping signal 20 TSTP  
07/22/2014 13:11:05 [500:43604]: mapped signal TSTP to signal KILL  
07/22/2014 13:11:05 [500:43604]: queued signal KILL  
07/22/2014 13:11:05 [500:43604]: /SGE/GE2011.11/inra/tools/terminate\_method.sh $job\_owner $job\_pid -\> overriddes kill(-43606, KILL)  
07/22/2014 13:11:05 [500:43604]: Skipped start of suspend: previous command (pid= 52460) is still active  
07/22/2014 13:11:06 [500:43604]: wait3 returned -1  
07/22/2014 13:11:06 [500:43604]: forward\_signal\_to\_job(): mapping signal 20 TSTP  
07/22/2014 13:11:06 [500:43604]: mapped signal TSTP to signal KILL  
07/22/2014 13:11:06 [500:43604]: queued signal KILL  
07/22/2014 13:11:06 [500:43604]: /SGE/GE2011.11/inra/tools/terminate\_method.sh $job\_owner $job\_pid -\> overriddes kill(-43606, KILL)  
07/22/2014 13:11:06 [500:43604]: Skipped start of suspend: previous command (pid= 52460) is still active  
07/22/2014 13:11:06 [500:43604]: wait3 returned 43606 (status: 15; WIFSIGNALED: 1, WIFEXITED: 0, WEXITSTATUS: 0)  
07/22/2014 13:11:06 [500:43604]: job exited with exit status 0  
07/22/2014 13:11:09 [500:43604]: wait3 returned 52460 (status: 0; WIFSIGNALED: 0, WIFEXITED: 1, WEXITSTATUS: 0)  
07/22/2014 13:11:09 [500:43604]: reaped terminate command  
07/22/2014 13:11:09 [500:43604]: reaped “job” with pid 43606  
07/22/2014 13:11:09 [500:43604]: job exited due to signal  
07/22/2014 13:11:09 [500:43604]: job signaled: 15  
07/22/2014 13:11:09 [500:43604]: now sending signal KILL to pid -43606  
07/22/2014 13:11:09 [500:43604]: writing usage file to “usage”  
07/22/2014 13:11:09 [500:43604]: no tasker to notify  
07/22/2014 13:11:09 [500:43604]: parent: forked “epilog” with pid 52620  
07/22/2014 13:11:09 [500:43604]: using signal delivery delay of 120 seconds  
07/22/2014 13:11:09 [500:43604]: parent: epilog-pid: 52620  
07/22/2014 13:11:09 [500:52620]: child: starting son(epilog, /SGE/GE2011.11/inra/tools/epilog\_verbose.sh, 0);  
07/22/2014 13:11:09 [500:52620]: pid=52620 pgrp=52620 sid=52620 old pgrp=43604 getlogin()=  
07/22/2014 13:11:09 [500:52620]: reading passwd information for user ‘jauguet’  
07/22/2014 13:11:09 [500:52620]: setting limits  
07/22/2014 13:11:09 [500:52620]: setting environment  
07/22/2014 13:11:09 [500:52620]: Initializing error file  
07/22/2014 13:11:09 [500:52620]: switching to intermediate/target user  
07/22/2014 13:11:09 [13532:52620]: closing all filedescriptors  
07/22/2014 13:11:09 [13532:52620]: further messages are in “error” and “trace”  
07/22/2014 13:11:09 [13532:52620]: using “/bin/bash” as shell of user “jauguet”  
07/22/2014 13:11:09 [13532:52620]: now running with uid=13532, euid=13532  
07/22/2014 13:11:09 [13532:52620]: execvp(/SGE/GE2011.11/inra/tools/epilog\_verbose.sh, “/SGE/GE2011.11/inra/tools/epilog\_verbose.sh”)  
ERROR :\_

  
  
The mothur logfile is as follow:

_mothur \> classify.seqs(fasta=arch.trim.contigs.good.unique.good.filter.unique.precluster.pick.abund.fasta, count=arch.trim.contigs.good.unique.good.filter.unique.precluster.uchime.pick.abund.count\_table, reference=gg\_99.pds.ng.fasta, taxonomy=gg\_99.pds.tax, cutoff=80, processors=12)_  
\_Using 12 processors.  
Generating search database… DONE.  
It took 89 seconds generate search database.

Reading in the gg\_99.pds.tax taxonomy… DONE.  
Calculating template taxonomy tree… DONE.  
Calculating template probabilities… DONE.  
It took 317 seconds get probabilities.  
Classifying sequences from arch.trim.contigs.good.unique.good.filter.unique.precluster.pick.abund.fasta …  
[WARNING]: M02233\_62\_000000000-A9GLW\_1\_1109\_23923\_13093 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.  
[WARNING]: M02233\_50\_000000000-A92MB\_1\_1105\_3231\_10647 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.  
[WARNING]: M02233\_50\_000000000-A92MB\_1\_1118\_12817\_9845 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.  
[WARNING]: M02233\_50\_000000000-A92MB\_1\_2118\_24109\_16291 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.  
[WARNING]: M02233\_50\_000000000-A92MB\_1\_2112\_6934\_15906 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.  
[WARNING]: M02233\_50\_000000000-A92MB\_1\_1104\_8028\_13594 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.  
[WARNING]: M02233\_62\_000000000-A9GLW\_1\_2117\_8803\_8563 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.  
[WARNING]: M02233\_62\_000000000-A9GLW\_1\_1106\_27288\_9106 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.  
[WARNING]: M02233\_50\_000000000-A92MB\_1\_2113\_14497\_12782 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.  
[WARNING]: M02233\_50\_000000000-A92MB\_1\_1104\_15585\_12202 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.  
[WARNING]: M02233\_62\_000000000-A9GLW\_1\_2117\_26720\_12683 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.  
[WARNING]: M02233\_50\_000000000-A92MB\_1\_2118\_16821\_22765 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.  
[WARNING]: M02233\_62\_000000000-A9GLW\_1\_2117\_26239\_9001 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.  
[WARNING]: M02233\_50\_000000000-A92MB\_1\_2112\_3567\_13138 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.  
[WARNING]: M02233\_50\_000000000-A92MB\_1\_1116\_10699\_11513 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.  
[WARNING]: M02233\_50\_000000000-A92MB\_1\_2118\_16255\_8301 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.  
[WARNING]: M02233\_50\_000000000-A92MB\_1\_2112\_17899\_18506 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.  
[WARNING]: M02233\_50\_000000000-A92MB\_1\_1103\_19986\_24223 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.  
[WARNING]: M02233\_50\_000000000-A92MB\_1\_2112\_17799\_24535 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.\_

I do not know if I’m doing something wrong but It worked like a charm with the Silva database. I precise that I work in batch mode with version 1.32.1. Sequences have been aligned with the greegenes database. The number of sequences should not be a problem since I have only 12120 unique sequences. Is the greengenes database so computational demanding that I have to increase the number of processors and RAM for the job?  
Thank in advance for the help.

JC

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<div class="post-metadata">

**Author:** ![pschloss](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/pschloss/32/4_2.png) [@pschloss](https://forum.mothur.org/u/pschloss)\
**Post date:** [July 24, 2014, 4:09pm UTC](https://forum.mothur.org/t/classify-seqs/1932/2 "2014-07-24T16:09:44Z")

</div>

A few things…

1. I’m not sure what’s going on with the “Unable to locate a module” error and I can’t make sense of why the scheduler killed it. You should check with your sys admin to see if they can translate what’s going on - that will help a lot. The greengenes database has 200k sequences in it whereas the silva database has 15k sequences. It’s not exactly a 10-fold increase in memory, but it is a bit more.

2. The warning messages just indicate what they say - it couldn’t classify the sequence to the kingdom level and so you should remove them afterwards with remove.lineage.

3. I’m not sure whether the reference files you are using are the same as the ones we have posted. Ours have names like gg\_13\_5\_99.pds\_gg.tax, gg\_13\_5\_99.fasta, and gg\_13\_5\_99.gg.tax

4. I don’t think it will matter, but you might try to upgrade to mothur v. 1.33

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<div class="post-metadata">

**Author:** ![jcauguet](https://avatars.discourse-cdn.com/v4/letter/j/b3f665/32.png) [@jcauguet](https://forum.mothur.org/u/jcauguet)\
**Post date:** [July 30, 2014, 12:00pm UTC](https://forum.mothur.org/t/classify-seqs/1932/3 "2014-07-30T12:00:06Z")

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Thanks Pat for your reply.  
Seems that it is a memory problem but I cannot stop thinking what would be good practice when the number of unique sequences is too high (for any reasons) for Miseq data and analyses are hampered by computational power (specially at the dist.seqs and cluster steps).  
All options (omitting phylotype analysis, but is it a valid option?) imply a reduction of the data set.  
One solution is subsampling before the dist.seq step but I’ve seen another approach (not completely sure it is correct) using the split.abund function with cutoff = 1 in order to remove singleton sequences. Am I wrong if i say that with this second option, I may get rid of (as an extreme example) abundant OTU’s regrouping these singleton sequences?  
From what I have read on this forum and others, we are quite a lot in having the same thoughts…  
Cheers

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<div class="post-metadata">

**Author:** ![pschloss](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/pschloss/32/4_2.png) [@pschloss](https://forum.mothur.org/u/pschloss)\
**Post date:** [July 30, 2014, 4:52pm UTC](https://forum.mothur.org/t/classify-seqs/1932/4 "2014-07-30T16:52:02Z")

</div>

For classify.seqs the memory problem is not related to the input dataset - it reads in one sequence at a time. The memory limitation is due to reading in the database.

Pat