# Bug in align.seqs? **URL:** \ **Category:** Commands in mothur\ **Created:** [January 15, 2015, 7:12pm UTC](https://forum.mothur.org/t/bug-in-align-seqs/2201 "2015-01-15T19:12:20Z")\ **Posts on this page:** 7\ **Page:** 1
**Author:** ![peleliu](https://avatars.discourse-cdn.com/v4/letter/p/8e7dd6/32.png) [@peleliu](https://forum.mothur.org/u/peleliu)\ **Post date:** [January 15, 2015, 7:12pm UTC](https://forum.mothur.org/t/bug-in-align-seqs/2201/1 "2015-01-15T19:12:20Z")
## Hi, I’m analyzing a full Miseq run (204 samples, ~50k reads per sample) on a linux server, following the Miseq SOP. It’s going great until the align.seqs, where the consequent summary.seqs is very weird and the # of unique seqs is wrong. Please help…! Logfile is attached: Linux version Using ReadLine Running 64Bit Version mothur v.1.34.4 Last updated: 12/22/2014 by Patrick D. Schloss Department of Microbiology & Immunology University of Michigan [pschloss@umich.edu](mailto:pschloss@umich.edu) [http://www.mothur.org](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 Type ‘quit()’ to exit program Interactive Mode mothur \> summary.seqs(fasta=stability.trim.contigs.good.fasta, processors=8) Using 8 processors. Start End NBases Ambigs Polymer NumSeqs Minimum: 1 250 250 0 3 1 2.5%-tile: 1 291 291 0 4 272820 25%-tile: 1 292 292 0 4 2728198 Median: 1 292 292 0 4 5456395 75%-tile: 1 292 292 0 5 8184592 97.5%-tile: 1 294 294 0 6 10639970 Maximum: 1 320 320 0 58 10912789 Mean: 1 292.112 292.112 0 4.71934 # of Seqs: 10912789 Output File Names: stability.trim.contigs.good.summary It took 70 secs to summarize 10912789 sequences. mothur \> screen.seqs(fasta=stability.trim.contigs.fasta, group=stability.contigs.groups, maxambig=0, maxlength=300) Using 8 processors. Output File Names: stability.trim.contigs.good.fasta stability.trim.contigs.bad.accnos stability.contigs.good.groups It took 282 secs to screen 16640727 sequences. mothur \> unique.seqs(fasta=stability.trim.contigs.good.fasta) 10911586 4549033 Output File Names: stability.trim.contigs.good.names stability.trim.contigs.good.unique.fasta mothur \> count.seqs(name=stability.trim.contigs.good.names, group=stability.contigs.good.groups) Using 8 processors. It took 284 secs to create a table for 10911586 sequences. Total number of sequences: 10911586 Output File Names: stability.trim.contigs.good.count\_table mothur \> summary.seqs(count=stability.trim.contigs.good.count\_table) Using stability.trim.contigs.good.unique.fasta as input file for the fasta parameter. Using 8 processors. Start End NBases Ambigs Polymer NumSeqs Minimum: 1 250 250 0 3 1 2.5%-tile: 1 291 291 0 4 272790 25%-tile: 1 292 292 0 4 2727897 Median: 1 292 292 0 4 5455794 75%-tile: 1 292 292 0 5 8183690 97.5%-tile: 1 294 294 0 6 10638797 Maximum: 1 300 300 0 58 10911586 Mean: 1 292.11 292.11 0 4.71927 # of unique seqs: 4549033 total # of seqs: 10911586 Output File Names: stability.trim.contigs.good.unique.summary It took 98 secs to summarize 10911586 sequences. mothur \> align.seqs(fasta=stability.trim.contigs.good.unique.fasta, reference=silva.bacteria.fasta) Using 8 processors. Reading in the silva.bacteria.fasta template sequences… DONE. It took 19 to read 14956 sequences. Aligning sequences from stability.trim.contigs.good.unique.fasta … Some of you sequences generated alignments that eliminated too many bases, a list is provided in stability.trim.contigs.good.unique.flip.accnos. If you set the flip parameter to true mothur will try aligning the reverse compliment as well. It took 15622 secs to align 4549033 sequences. Output File Names: stability.trim.contigs.good.unique.align stability.trim.contigs.good.unique.align.report stability.trim.contigs.good.unique.flip.accnos mothur \> summary.seqs(fasta=stability.trim.contigs.good.unique.align, count=stability.trim.contigs.good.count\_table) Using 8 processors. Start End NBases Ambigs Polymer NumSeqs Minimum: -1 -1 0 0 1 1 2.5%-tile: 11895 25318 292 0 4 272790 25%-tile: 43116 43116 300 0 46 2727897 Median: 0 0 0 0 0 5455794 75%-tile: 0 0 0 0 0 8183690 97.5%-tile: 0 0 0 0 0 10638797 Maximum: 43116 43116 300 0 46 10911586 Mean: 2907.94 6188.81 71.399 0 1.20989 # of unique seqs: 630790 total # of seqs: 10911586 Output File Names: stability.trim.contigs.good.unique.summary It took 566 secs to summarize 10911586 sequences. mothur \> summary.seqs(fasta=stability.trim.contigs.good.unique.align, count=stability.trim.contigs.good.count\_table) Using 8 processors. Start End NBases Ambigs Polymer NumSeqs Minimum: -1 -1 0 0 1 1 2.5%-tile: 11895 25318 292 0 4 272790 25%-tile: 43116 43116 300 0 46 2727897 Median: 0 0 0 0 0 5455794 75%-tile: 0 0 0 0 0 8183690 97.5%-tile: 0 0 0 0 0 10638797 Maximum: 43116 43116 300 0 46 10911586 Mean: 2920.48 6208.38 71.5522 0 1.2124 # of unique seqs: 634493 total # of seqs: 10911586 Output File Names: stability.trim.contigs.good.unique.summary It took 570 secs to summarize 10911586 sequences. mothur \> summary.seqs(count=stability.trim.contigs.good.count\_table) Using stability.trim.contigs.good.unique.align as input file for the fasta parameter. Using 8 processors. Start End NBases Ambigs Polymer NumSeqs Minimum: -1 -1 0 0 1 1 2.5%-tile: 11895 25318 292 0 4 272790 25%-tile: 43116 43116 300 0 46 2727897 Median: 0 0 0 0 0 5455794 75%-tile: 0 0 0 0 0 8183690 97.5%-tile: 0 0 0 0 0 10638797 Maximum: 43116 43116 300 0 46 10911586 Mean: 2920.48 6208.38 71.5522 0 1.2124 # of unique seqs: 634493 total # of seqs: 10911586 Output File Names: stability.trim.contigs.good.unique.summary It took 570 secs to summarize 10911586 sequences. mothur \> summary.seqs(fasta=stability.trim.contigs.good.unique.align, count=stability.trim.contigs.good.count\_table, processors=1) Using 1 processors. Start End NBases Ambigs Polymer NumSeqs Minimum: -1 -1 0 0 1 1 2.5%-tile: 11895 25318 292 0 4 272790 25%-tile: 43116 43116 300 0 46 2727897 Median: 0 0 0 0 0 5455794 75%-tile: 0 0 0 0 0 8183690 97.5%-tile: 0 0 0 0 0 10638797 Maximum: 43116 43116 300 0 46 10911586 Mean: 2920.48 6208.38 71.5522 0 1.2124 # of unique seqs: 634493 total # of seqs: 10911586 Output File Names: stability.trim.contigs.good.unique.summary It took 881 secs to summarize 10911586 sequences. ---
**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:** [January 16, 2015, 4:20pm UTC](https://forum.mothur.org/t/bug-in-align-seqs/2201/2 "2015-01-16T16:20:36Z")
Could you delete silva.bacteria.fasta and the other silva.bactera like files, redownload them and try align.seqs again? Also, are you sure that you put the R1 and R2 files names in the correct columns of your files file? pat ---
**Author:** ![peleliu](https://avatars.discourse-cdn.com/v4/letter/p/8e7dd6/32.png) [@peleliu](https://forum.mothur.org/u/peleliu)\ **Post date:** [January 18, 2015, 1:06pm UTC](https://forum.mothur.org/t/bug-in-align-seqs/2201/3 "2015-01-18T13:06:10Z")
Pat, Thanks for the reply. I re-downloaded silva.bacteria.fasta, and now Mothur can’t even read it, I get an error message saying that the template (i.e. new reference file) is not aligned. I get the same error message when trying align.seqs with the silva seed\_v119.align. P. ---
**Author:** ![westcott](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/westcott/32/18_2.png) [@westcott](https://forum.mothur.org/u/westcott)\ **Post date:** [January 21, 2015, 4:33pm UTC](https://forum.mothur.org/t/bug-in-align-seqs/2201/4 "2015-01-21T16:33:03Z")
I am not able to reproduce the problems you are having on my Linux test machine with our executable. Are you using the executable version of mothur or did you build from source? ---
**Author:** ![peleliu](https://avatars.discourse-cdn.com/v4/letter/p/8e7dd6/32.png) [@peleliu](https://forum.mothur.org/u/peleliu)\ **Post date:** [January 21, 2015, 8:13pm UTC](https://forum.mothur.org/t/bug-in-align-seqs/2201/5 "2015-01-21T20:13:06Z")
Executable version, linux 64 on Amazon EC2. ---
**Author:** ![westcott](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/westcott/32/18_2.png) [@westcott](https://forum.mothur.org/u/westcott)\ **Post date:** [January 22, 2015, 8:19pm UTC](https://forum.mothur.org/t/bug-in-align-seqs/2201/6 "2015-01-22T20:19:27Z")
Could you send your fasta file to [mothur.bugs@gmail.com](mailto:mothur.bugs@gmail.com)? ---
**Author:** ![peleliu](https://avatars.discourse-cdn.com/v4/letter/p/8e7dd6/32.png) [@peleliu](https://forum.mothur.org/u/peleliu)\ **Post date:** [February 1, 2015, 5:38pm UTC](https://forum.mothur.org/t/bug-in-align-seqs/2201/7 "2015-02-01T17:38:48Z")
Sent it last week 🙂 Thanks for the help!