# The align.seqs command is returning a blank file **URL:** \ **Category:** Commands in mothur\ **Created:** [March 8, 2022, 5:52pm UTC](https://forum.mothur.org/t/the-align-seqs-command-is-returning-a-blank-file/21327 "2022-03-08T17:52:55Z")\ **Posts on this page:** 6\ **Page:** 1
**Author:** ![jfulgini](https://avatars.discourse-cdn.com/v4/letter/j/ee59a6/32.png) [@jfulgini](https://forum.mothur.org/u/jfulgini)\ **Post date:** [March 8, 2022, 5:52pm UTC](https://forum.mothur.org/t/the-align-seqs-command-is-returning-a-blank-file/21327/1 "2022-03-08T17:52:55Z")
Hello mothur community. I’m a grad student who is being introduced to bioinformatics, so my familiarity with troubleshooting mothur issues is not the best right now… About a fifth of my samples (reps included) are returning blank align files after I run the align.seqs command, and I’m not too sure why given that the script of code I have works for the majority of my samples. I attached my logfile in the text below, hopefully that will give you guys enough information to work with. The summary.seqs command I used prior to mothur telling me my align file was blank was not included in the logfile. It should also be noted that mothur crashed when I tried to run the command too. ```auto mothur > list.seqs(fastq=ETN.20.KN14.swab.3.2_R2.fastq) Output File Names: ETN.20.KN14.swab.3.2_R2.accnos mothur > get.seqs(fastq=ETN.20.KN14.swab.3.2_R1.fastq, accnos=ETN.20.KN14.swab.3.2_R2.accnos) Selected 243126 sequences from ETN.20.KN14.swab.3.2_R1.fastq. Output File Names: ETN.20.KN14.swab.3.2_R1.pick.fastq mothur > list.seqs(fastq=ETN.20.KN14.swab.3.2_R1.pick.fastq) Output File Names: ETN.20.KN14.swab.3.2_R1.pick.accnos mothur > get.seqs(fastq=ETN.20.KN14.swab.3.2_R2.fastq, accnos=ETN.20.KN14.swab.3.2_R1.pick.accnos) Selected 243126 sequences from ETN.20.KN14.swab.3.2_R2.fastq. Output File Names: ETN.20.KN14.swab.3.2_R2.pick.fastq mothur > make.contigs(ffastq = ETN.20.KN14.swab.3.2_R1.pick.fastq, rfastq = ETN.20.KN14.swab.3.2_R2.pick.fastq, trimoverlap =T, insert = 24, deltaq =6, proccessors=1) [WARNING]: proccessors is not a valid parameter, ignoring. The valid parameters are: ffastq, rfastq, ffasta, rfasta, fqfile, rqfile, file, oligos, findex, rindex, qfile, pdiffs, bdiffs, tdiffs, checkorient, align, allfiles, trimoverlap, match, mismatch, gapopen, gapextend, insert, deltaq, maxee, processors, format, ksize, maxambig, maxhomop, maxlength, seed, inputdir, and outputdir. Using 8 processors. Making contigs... Done. It took 32 secs to process 243126 sequences. Output File Names: ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.fasta ETN.20.KN14.swab.3.2_R1.pick.scrap.contigs.fasta ETN.20.KN14.swab.3.2_R1.pick.contigs_report mothur > summary.seqs(fasta= ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.fasta) Using 8 processors. Start End NBases Ambigs Polymer NumSeqs Minimum: 1 1 1 0 1 1 2.5%-tile: 1 201 201 0 3 6078 25%-tile: 1 202 202 0 4 60774 Median: 1 202 202 0 4 121548 75%-tile: 1 202 202 0 4 182321 97.5%-tile: 1 207 207 1 5 237017 Maximum: 1 217 217 53 11 243094 Mean: 1 201 201 0 4 # of Seqs: 243094 It took 1 secs to summarize 243094 sequences. Output File Names: ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.summary mothur > make.group(fasta=ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.fasta, groups=ETN.20.KN14.swab.3.2.W, output=ETN.20.KN14.swab.3.2.groups) Output File Names: ETN.20.KN14.swab.3.2.groups mothur > screen.seqs(fasta=ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.fasta, group=ETN.20.KN14.swab.3.2.groups, maxambig= 0, maxhomop=8) Using 8 processors. It took 0 secs to screen 243094 sequences, removed 13074. / ****************************************** / Running command: remove.seqs(accnos=ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.bad.accnos.temp, group=ETN.20.KN14.swab.3.2.groups) Removed 13074 sequences from ETN.20.KN14.swab.3.2.groups. Output File Names: ETN.20.KN14.swab.3.2.pick.groups / ****************************************** / Output File Names: ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.good.fasta ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.bad.accnos ETN.20.KN14.swab.3.2.good.groups It took 2 secs to screen 243094 sequences. mothur > summary.seqs(fasta = ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.good.fasta) Using 8 processors. Start End NBases Ambigs Polymer NumSeqs Minimum: 1 10 10 0 2 1 2.5%-tile: 1 201 201 0 3 5751 25%-tile: 1 202 202 0 4 57506 Median: 1 202 202 0 4 115011 75%-tile: 1 202 202 0 4 172516 97.5%-tile: 1 206 206 0 5 224270 Maximum: 1 217 217 0 8 230020 Mean: 1 202 202 0 4 # of Seqs: 230020 It took 1 secs to summarize 230020 sequences. Output File Names: ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.good.summary mothur > [ERROR]: You are missing ( [ERROR]: Invalid. mothur > unique.seqs(fasta = ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.good.fasta) 230020 44523 Output File Names: ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.good.unique.fasta ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.good.count_table mothur > summary.seqs(fasta=ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.good.unique.fasta, count=ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.good.count_table) Using 8 processors. Start End NBases Ambigs Polymer NumSeqs Minimum: 1 10 10 0 2 1 2.5%-tile: 1 201 201 0 3 5751 25%-tile: 1 202 202 0 4 57506 Median: 1 202 202 0 4 115011 75%-tile: 1 202 202 0 4 172516 97.5%-tile: 1 206 206 0 5 224270 Maximum: 1 217 217 0 8 230020 Mean: 1 202 202 0 4 # of unique seqs: 44523 total # of seqs: 230020 It took 1 secs to summarize 230020 sequences. Output File Names: ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.good.unique.summary mothur > align.seqs(fasta= ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.good.unique.fasta, reference= silva.nr_v138_1.align) Using 8 processors. Reading in the silva.nr_v138_1.align template sequences... DONE. It took 156 to read 146601 sequences. Aligning sequences from ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.good.unique.fasta ... It took 109 secs to align 44523 sequences. It took 117 seconds to align 44523 sequences. Output File Names: ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.good.unique.align ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.good.unique.align_report ``` ---
**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:** [March 8, 2022, 7:42pm UTC](https://forum.mothur.org/t/the-align-seqs-command-is-returning-a-blank-file/21327/2 "2022-03-08T19:42:19Z")
Hi there, What region of the 16S rRNA gene are you sequencing? What version number of mothur and what operating system are you using? Pat ---
**Author:** ![jfulgini](https://avatars.discourse-cdn.com/v4/letter/j/ee59a6/32.png) [@jfulgini](https://forum.mothur.org/u/jfulgini)\ **Post date:** [March 8, 2022, 8:11pm UTC](https://forum.mothur.org/t/the-align-seqs-command-is-returning-a-blank-file/21327/3 "2022-03-08T20:11:50Z")
We sequenced the V4 region, we’re using mothur v.1.47 ., and we’re using windows 7. ---
**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:** [March 9, 2022, 3:35pm UTC](https://forum.mothur.org/t/the-align-seqs-command-is-returning-a-blank-file/21327/4 "2022-03-09T15:35:03Z")
Could you send your fasta file to [mothur.bugs@gmail.com](mailto:mothur.bugs@gmail.com) so I can take a closer look? Also, mothur is built for Windows 10. There may be unexpected behavior running it on Windows 7 (release date 2009, support ended 2020). Can you upgrade your version of Windows? ---
**Author:** ![jfulgini](https://avatars.discourse-cdn.com/v4/letter/j/ee59a6/32.png) [@jfulgini](https://forum.mothur.org/u/jfulgini)\ **Post date:** [March 11, 2022, 4:11pm UTC](https://forum.mothur.org/t/the-align-seqs-command-is-returning-a-blank-file/21327/5 "2022-03-11T16:11:49Z")
I apologize, sending them to mothur bugs right now ---
**Author:** ![system](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/system/32/2_2.png) [@system](https://forum.mothur.org/u/system)\ **Post date:** [March 21, 2022, 4:12pm UTC](https://forum.mothur.org/t/the-align-seqs-command-is-returning-a-blank-file/21327/6 "2022-03-21T16:12:21Z")
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