# Pcr.seqs \[ERROR\]: seqs are not aligned. When using start and end your sequences must be aligned **URL:** \ **Category:** Commands in mothur\ **Created:** [September 30, 2021, 7:59pm UTC](https://forum.mothur.org/t/pcr-seqs-error-seqs-are-not-aligned-when-using-start-and-end-your-sequences-must-be-aligned/21147 "2021-09-30T19:59:04Z")\ **Posts on this page:** 10\ **Page:** 1
**Author:** ![ADL](https://avatars.discourse-cdn.com/v4/letter/a/3ec8ea/32.png) [@ADL](https://forum.mothur.org/u/ADL)\ **Post date:** [September 30, 2021, 7:59pm UTC](https://forum.mothur.org/t/pcr-seqs-error-seqs-are-not-aligned-when-using-start-and-end-your-sequences-must-be-aligned/21147/1 "2021-09-30T19:59:04Z")
I am using Mothur version 1.46.1, within Ubuntu terminal for Windows10 I ran align.seqs with the silva138 reference alignment and the rrnDB fasta file align.seqs(fasta=data/raw/rrnDB-5.7\_16S\_rRNA.fasta, reference=data/references/silva\_seed/silva.seed\_v138.align, flip=T) This ran as expected, however pcr.seqs on the resulting .align file gave the error: pcr.seqs(fasta=data/raw/rrnDB-5.7\_16S\_rRNA.align, start=1044, end=43116, outputdir=data/v19/) [ERROR]: seqs are not aligned. When using start and end your sequences must be aligned. I re-downloaded all the files and tried again with the same result ---
**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:** [October 5, 2021, 4:39pm UTC](https://forum.mothur.org/t/pcr-seqs-error-seqs-are-not-aligned-when-using-start-and-end-your-sequences-must-be-aligned/21147/2 "2021-10-05T16:39:10Z")
Hi ADL, Can you post the output of running… ```auto summary.seqs(fasta=data/raw/rrnDB-5.7_16S_rRNA.align) ``` Thanks, Pat ---
**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:** [October 5, 2021, 4:46pm UTC](https://forum.mothur.org/t/pcr-seqs-error-seqs-are-not-aligned-when-using-start-and-end-your-sequences-must-be-aligned/21147/3 "2021-10-05T16:46:22Z")
Could you send the rrnDB-5.7\_16S\_rRNA.fasta file to [mothur.bugs@gmail.com](mailto:mothur.bugs@gmail.com) so I can take a closer look for you? ---
**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:** [October 6, 2021, 6:08pm UTC](https://forum.mothur.org/t/pcr-seqs-error-seqs-are-not-aligned-when-using-start-and-end-your-sequences-must-be-aligned/21147/4 "2021-10-06T18:08:21Z")
Have you tried running mothur natively on your Windows machine, [https://github.com/mothur/mothur/releases/download/v1.46.1/Mothur.win.zip](https://github.com/mothur/mothur/releases/download/v1.46.1/Mothur.win.zip)? ---
**Author:** ![ADL](https://avatars.discourse-cdn.com/v4/letter/a/3ec8ea/32.png) [@ADL](https://forum.mothur.org/u/ADL)\ **Post date:** [October 7, 2021, 2:51pm UTC](https://forum.mothur.org/t/pcr-seqs-error-seqs-are-not-aligned-when-using-start-and-end-your-sequences-must-be-aligned/21147/5 "2021-10-07T14:51:34Z")
`mothur > summary.seqs(fasta=data/raw/rrnDB-5.7_16S_rRNA.align)` ```auto Using 8 processors. Start End NBases Ambigs Polymer NumSeqs Minimum: 1044 16429 569 0 4 1 2.5%-tile: 1044 43116 1401 0 5 230 25%-tile: 1044 43116 1437 0 5 2291 Median: 1044 43116 1464 0 6 4582 75%-tile: 1044 43116 1464 0 6 6873 97.5%-tile: 1044 43116 1475 0 7 8934 Maximum: 7922 43116 1576 4 9 9163 Mean: 1048 43111 1453 0 5 # of Seqs: 9163 It took 14 secs to summarize 9163 sequences. Output File Names: data/raw/rrnDB-5.7_16S_rRNA.summary ``` ---
**Author:** ![ADL](https://avatars.discourse-cdn.com/v4/letter/a/3ec8ea/32.png) [@ADL](https://forum.mothur.org/u/ADL)\ **Post date:** [October 7, 2021, 2:52pm UTC](https://forum.mothur.org/t/pcr-seqs-error-seqs-are-not-aligned-when-using-start-and-end-your-sequences-must-be-aligned/21147/6 "2021-10-07T14:52:18Z")
Sorry, the formatting is bad. But they sure look aligned ---
**Author:** ![ADL](https://avatars.discourse-cdn.com/v4/letter/a/3ec8ea/32.png) [@ADL](https://forum.mothur.org/u/ADL)\ **Post date:** [October 8, 2021, 6:41pm UTC](https://forum.mothur.org/t/pcr-seqs-error-seqs-are-not-aligned-when-using-start-and-end-your-sequences-must-be-aligned/21147/7 "2021-10-08T18:41:20Z")
Ok, I’m running everything again with the most recent rrnDB files (5.7) and mothur version and now it appears to be working. I did nothing different. Every time before, the command for pcr.seqs terminated after a few seconds, with the error that the .align was not aligned. So I suppose my issue is resolved, but I’m not sure why. ---
**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:** [October 12, 2021, 1:39pm UTC](https://forum.mothur.org/t/pcr-seqs-error-seqs-are-not-aligned-when-using-start-and-end-your-sequences-must-be-aligned/21147/8 "2021-10-12T13:39:43Z")
Thanks for the update. Just to clarify, is it working using mothur’s windows version or are you still using the Ubuntu terminal on Windows? ---
**Author:** ![ADL](https://avatars.discourse-cdn.com/v4/letter/a/3ec8ea/32.png) [@ADL](https://forum.mothur.org/u/ADL)\ **Post date:** [October 12, 2021, 1:52pm UTC](https://forum.mothur.org/t/pcr-seqs-error-seqs-are-not-aligned-when-using-start-and-end-your-sequences-must-be-aligned/21147/9 "2021-10-12T13:52:29Z")
It’s working on the Ubuntu terminal ---
**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:** [October 22, 2021, 1:52pm UTC](https://forum.mothur.org/t/pcr-seqs-error-seqs-are-not-aligned-when-using-start-and-end-your-sequences-must-be-aligned/21147/10 "2021-10-22T13:52:53Z")
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