# Fragment insertion SEPP issue
**URL:** https://forum.qiime2.org/t/fragment-insertion-sepp-issue/6482
**Category:** User Support
**Created:** [October 17, 2018, 5:11am UTC](https://forum.qiime2.org/t/fragment-insertion-sepp-issue/6482 "2018-10-17T05:11:24Z")
**Posts on this page:** 7
**Page:** 1
### Author:  [@Fra](https://forum.qiime2.org/u/Fra)
#### Post date: [October 17, 2018, 5:11am UTC](https://forum.qiime2.org/t/fragment-insertion-sepp-issue/6482/1 "2018-10-17T05:11:24Z")
Hello everybody,
I have 4 16S datasets, 3 of these use region V4 and one another region, so I run the cmd:
qiime fragment-insertion sepp --i-representative-sequences 16S\_repr\_seq\_deblur\_merged\_run.qza --o-tree 16S\_insertion\_tree.qza --o-placements 16S\_rinsertion\_placement.qza --p-threads 16 --verbose
And I get this errors a pile of errors of these nature:
alignment.py (line 532): INFO: Merging extension sto file (/media/nec3\_HDD/Francesco/tmp/tmp.Aa2TqJRZnK/sepp-tempssd-sxeg/q2-fragment-insertion.9xptp35d/root/P\_55/A\_55\_3/FC\_55\_3\_3/hmmalign.results.\_qq\_7bi0) into base alignment (/media/nec3\_HDD/Francesco/tmp/tmp.Aa2TqJRZnK/sepp-tempssd-sxeg/q2-fragment-insertion.9xptp35d/root/P\_55/A\_55\_3/hmmbuild.input.kzhvovca.fasta).
jobs.py (line 117): INFO: Finished pplacer Job with input: backbone\_alignment\_file:/media/nec3\_HDD/Francesco/tmp/tmp.Aa2TqJRZnK/sepp-tempssd-sxeg/q2-fragment-insertion.9xptp35d/root/P\_33/pplacer.backbone.ytqgdbx1.fasta, tree\_file:/media/nec3\_HDD/Francesco/tmp/tmp.Aa2TqJRZnK/sepp-tempssd-sxeg/q2-fragment-insertion.9xptp35d/root/P\_33/pplacer.tree.40l9lt1m.tre, info\_file:/media/nec3\_HDD/miniconda3/envs/qiime2-2018.8/share/fragment-insertion//ref/RAxML\_info-reference-gg-raxml-bl.info, extended alignment:/media/nec3\_HDD/Francesco/tmp/tmp.Aa2TqJRZnK/sepp-tempssd-sxeg/q2-fragment-insertion.9xptp35d/root/P\_33/pplacer.extended.0.ce07e66a.fasta, output:/media/nec3\_HDD/Francesco/tmp/tmp.Aa2TqJRZnK/sepp-tempssd-sxeg/q2-fragment-insertion.9xptp35d/root/P\_33/pplacer.extended.0.ce07e66a.jplace
Thanks for your help!
---
### Author:  [@Fra](https://forum.qiime2.org/u/Fra)
#### Post date: [October 17, 2018, 7:03am UTC](https://forum.qiime2.org/t/fragment-insertion-sepp-issue/6482/2 "2018-10-17T07:03:44Z")
If I run this cmd using only two datasets it works, one V4 dataset and the other V region.
---
### Author:  [@Nicholas\_Bokulich](https://forum.qiime2.org/u/Nicholas_Bokulich)
#### Post date: [October 17, 2018, 12:43pm UTC](https://forum.qiime2.org/t/fragment-insertion-sepp-issue/6482/3 "2018-10-17T12:43:20Z")
Thanks for reporting @Fra! I am cc:ing @Stefan to take a look at this.
---
### Author:  [@Stefan](https://forum.qiime2.org/u/Stefan)
#### Post date: [October 17, 2018, 1:16pm UTC](https://forum.qiime2.org/t/fragment-insertion-sepp-issue/6482/4 "2018-10-17T13:16:35Z")
Hi Francesco,
would you mind mailing me your input data to [stefan.m.janssen@gmail.com](mailto:stefan.m.janssen@gmail.com) ?
The "errors" you pasted are only debugging information. I cannot see if it really is an error :-/
You could also run the same command with additional parameter in the hope to shed more light on this issue: `--p-debug --verbose`
---
### Author:  [@Fra](https://forum.qiime2.org/u/Fra)
#### Post date: [October 17, 2018, 10:33pm UTC](https://forum.qiime2.org/t/fragment-insertion-sepp-issue/6482/5 "2018-10-17T22:33:46Z")
H Stefan,
I actually run the command again and this time worked. However, at the end of the pipeline after checking the PCoA the 4 different runs still group by primer. Basically even though the script produced an output it did not do its work properly.
Do you have any clue?
Cheers
---
### Author:  [@ebolyen](https://forum.qiime2.org/u/ebolyen)
#### Post date: [October 19, 2018, 8:16pm UTC](https://forum.qiime2.org/t/fragment-insertion-sepp-issue/6482/6 "2018-10-19T20:16:34Z")
Hi @Fra,
What metric did you use? It sounds like you have a batch effect at this point and if you used a quantitative metric in your PCoA that would only exacerbate the issue.
---
### Author:  [@system](https://forum.qiime2.org/u/system)
#### Post date: [November 20, 2018, 2:16am UTC](https://forum.qiime2.org/t/fragment-insertion-sepp-issue/6482/7 "2018-11-20T02:16:40Z")
This topic was automatically closed 31 days after the last reply. New replies are no longer allowed.