# Error whilst using v-search join pairs **URL:** \ **Category:** Technical Support\ **Created:** [November 29, 2019, 4:28am UTC](https://forum.qiime2.org/t/error-whilst-using-v-search-join-pairs/12667 "2019-11-29T04:28:24Z")\ **Posts on this page:** 9\ **Page:** 1
**Author:** ![lghuggins](https://forum.qiime2.org/letter_avatar_proxy/v4/letter/l/ba8739/32.png) [@lghuggins](https://forum.qiime2.org/u/lghuggins)\ **Post date:** [November 29, 2019, 4:28am UTC](https://forum.qiime2.org/t/error-whilst-using-v-search-join-pairs/12667/1 "2019-11-29T04:28:24Z")
I am attempting to join paired end reads using the v-search join pairs tool so that I can later perform Deblur to filter and clean up my data. I am doing 16S rRNA sequencing on a ~450bp amplified region. The command I am running is qiime vsearch join-pairs --i-demultiplexed-seqs Gya-cambodia-bacteria-paired-end-demux2.qza --o-joined-sequences Gya-cambodia-bacteria-paired-end-demux-joined2.qza --verbose and I get the error message: /home/lucas/miniconda3/envs/qiime2-2019.7/lib/python3.6/site-packages/q2\_vsearch/\_join\_pairs.py:90: YAMLLoadWarning: calling yaml.load() without Loader=... is deprecated, as the default Loader is unsafe. Please read [https://msg.pyyaml.org/load](https://msg.pyyaml.org/load) for full details. demultiplexed\_seqs.metadata.pathspec)))['phred-offset'] Running external command line application. This may print messages to stdout and/or stderr. The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist. Command: vsearch --fastq\_mergepairs /home/Public/Ps2/PsTmp/qiime2-archive-crik8\_du/db07cc02-bfd6-429f-9655-49d098f7ad08/data/1-GYA\_0\_L001\_R1\_001.fastq.gz --reverse /home/Public/Ps2/PsTmp/qiime2-archive-crik8\_du/db07cc02-bfd6-429f-9655-49d098f7ad08/data/1-GYA\_1\_L001\_R2\_001.fastq.gz --fastqout /home/Public/Ps2/PsTmp/q2-SingleLanePerSampleSingleEndFastqDirFmt-r9u86guo/1-GYA\_0\_L001\_R1\_001.fastq --fastq\_ascii 33 --fastq\_minlen 1 --fastq\_minovlen 10 --fastq\_maxdiffs 10 --fastq\_qmin 0 --fastq\_qminout 0 --fastq\_qmax 41 --fastq\_qmaxout 41 vsearch v2.7.0\_linux\_x86\_64, 141.5GB RAM, 16 cores > **[GitHub - torognes/vsearch: Versatile open-source tool for microbiome analysis](https://github.com/torognes/vsearch)** > > Versatile open-source tool for microbiome analysis Merging reads 100% Fatal error: More forward reads than reverse reads Traceback (most recent call last): File "/home/lucas/miniconda3/envs/qiime2-2019.7/lib/python3.6/site-packages/q2cli/commands.py", line 327, in **call** results = action(\*\*arguments) File "\", line 2, in join\_pairs File "/home/lucas/miniconda3/envs/qiime2-2019.7/lib/python3.6/site-packages/qiime2/sdk/action.py", line 240, in bound\_callable output\_types, provenance) File "/home/lucas/miniconda3/envs/qiime2-2019.7/lib/python3.6/site-packages/qiime2/sdk/action.py", line 383, in _callable\_executor_ output\_views = self.\_callable(\*\*view\_args) File "/home/lucas/miniconda3/envs/qiime2-2019.7/lib/python3.6/site-packages/q2\_vsearch/\_join\_pairs.py", line 57, in join\_pairs qmax, qmaxout) File "/home/lucas/miniconda3/envs/qiime2-2019.7/lib/python3.6/site-packages/q2\_vsearch/\_join\_pairs.py", line 141, in \_join\_pairs\_w\_command\_output run\_command(cmd) File "/home/lucas/miniconda3/envs/qiime2-2019.7/lib/python3.6/site-packages/q2\_vsearch/\_cluster\_features.py", line 33, in run\_command subprocess.run(cmd, check=True) File "/home/lucas/miniconda3/envs/qiime2-2019.7/lib/python3.6/subprocess.py", line 418, in run output=stdout, stderr=stderr) subprocess.CalledProcessError: Command '['vsearch', '--fastq\_mergepairs', '/home/Public/Ps2/PsTmp/qiime2-archive-crik8\_du/db07cc02-bfd6-429f-9655-49d098f7ad08/data/1-GYA\_0\_L001\_R1\_001.fastq.gz', '--reverse', '/home/Public/Ps2/PsTmp/qiime2-archive-crik8\_du/db07cc02-bfd6-429f-9655-49d098f7ad08/data/1-GYA\_1\_L001\_R2\_001.fastq.gz', '--fastqout', '/home/Public/Ps2/PsTmp/q2-SingleLanePerSampleSingleEndFastqDirFmt-r9u86guo/1-GYA\_0\_L001\_R1\_001.fastq', '--fastq\_ascii', '33', '--fastq\_minlen', '1', '--fastq\_minovlen', '10', '--fastq\_maxdiffs', '10', '--fastq\_qmin', '0', '--fastq\_qminout', '0', '--fastq\_qmax', '41', '--fastq\_qmaxout', '41']' returned non-zero exit status 1. Plugin error from vsearch: Command '['vsearch', '--fastq\_mergepairs', '/home/Public/Ps2/PsTmp/qiime2-archive-crik8\_du/db07cc02-bfd6-429f-9655-49d098f7ad08/data/1-GYA\_0\_L001\_R1\_001.fastq.gz', '--reverse', '/home/Public/Ps2/PsTmp/qiime2-archive-crik8\_du/db07cc02-bfd6-429f-9655-49d098f7ad08/data/1-GYA\_1\_L001\_R2\_001.fastq.gz', '--fastqout', '/home/Public/Ps2/PsTmp/q2-SingleLanePerSampleSingleEndFastqDirFmt-r9u86guo/1-GYA\_0\_L001\_R1\_001.fastq', '--fastq\_ascii', '33', '--fastq\_minlen', '1', '--fastq\_minovlen', '10', '--fastq\_maxdiffs', '10', '--fastq\_qmin', '0', '--fastq\_qminout', '0', '--fastq\_qmax', '41', '--fastq\_qmaxout', '41']' returned non-zero exit status 1. See above for debug info. I am running qiime2-2019.7, through conda. As a test to check that there is enough overlap between forward and reverse reads, for one of my indexes I uploaded all the data into Geneious and was able to successfully pair and merge forward and reverse reads into ~450bp long sequences. I have tried to solve this issue using advice from similar posts but to no avail, any help would be greatly appreciated. ---
**Author:** ![jwdebelius](https://forum.qiime2.org/user_avatar/forum.qiime2.org/jwdebelius/32/9655_2.png) [@jwdebelius](https://forum.qiime2.org/u/jwdebelius)\ **Post date:** [November 29, 2019, 9:19am UTC](https://forum.qiime2.org/t/error-whilst-using-v-search-join-pairs/12667/2 "2019-11-29T09:19:41Z")
Hi @lghuggins, This is a guess, but are these the sequences from you rother post? > [@DADA2 fails to run due to 'Error in isBimeraDenovoTable'](https://forum.qiime2.org/t/dada2-fails-to-run-due-to-error-in-isbimeradenovotable/12344/3): > > Hi Nicholas, Thank you for help with this, the issue was whilst using a manifest to import the data, the R2 reads were automatically changed to R1s when transferring the manifest data from a spreadsheet, hence I was only importing the forward reads. Thanks again! You might try setting your `--p-truncqual` which may trim your reads, but would get rid of the last 50 or so bases that seem to have issues. Best, Justine ---
**Author:** ![jwdebelius](https://forum.qiime2.org/user_avatar/forum.qiime2.org/jwdebelius/32/9655_2.png) [@jwdebelius](https://forum.qiime2.org/u/jwdebelius)\ **Post date:** [November 29, 2019, 11:33am UTC](https://forum.qiime2.org/t/error-whilst-using-v-search-join-pairs/12667/5 "2019-11-29T11:33:17Z")
ETA: You might also want to check whether the forward and reverse sequences > [@lghuggins](#): > > Fatal error: More forward reads than reverse reads Best, Justine ---
**Author:** ![lghuggins](https://forum.qiime2.org/letter_avatar_proxy/v4/letter/l/ba8739/32.png) [@lghuggins](https://forum.qiime2.org/u/lghuggins)\ **Post date:** [December 2, 2019, 12:37am UTC](https://forum.qiime2.org/t/error-whilst-using-v-search-join-pairs/12667/6 "2019-12-02T00:37:35Z")
> [@jwdebelius](#): > > truncqual Hi @jwdebelius, Thanks for your advice, the sequences this time actually aren't from my previous post and I've rechecked to ensure that the imported data is separate forward and reverse reads which it is. See data quality plots here: ![image](https://forum-qiime2-org.s3.dualstack.us-west-2.amazonaws.com/original/2X/e/e6bbf89182d334cf5e79fdd3b09fb5b1042014d6.png) Before trying to join data using v-search I was attempting to join pairs and filter using DADA2 which I could not get to work either. Truncating reads to a variety of different lengths (both forward and reverse) including 200, 220, 240, 260 and 280 all failed to pair using DADA2. Would this not have had the same effect as the --p-truncqual command? If not which principal command would I run to complete --p-truncqual? V-search? Looking at other posts that have had a similar issue to mine having more forward than reverse reads as alluded to by this message: Fatal error: More forward reads than reverse reads Is not actually the issue as there are almost never the same number of forward and reverse reads, when I've done v-search join pairs and DADA2 commands before the difference between numbers of forward and reverse reads can be within the range of thousands and the command still runs, so I'm not sure this is the true problem behind my command failing. Any more help with this would be greatly appreciated. I've been able to analyse the data as single-end reads thus far. All the best, Lucas ---
**Author:** ![jwdebelius](https://forum.qiime2.org/user_avatar/forum.qiime2.org/jwdebelius/32/9655_2.png) [@jwdebelius](https://forum.qiime2.org/u/jwdebelius)\ **Post date:** [December 2, 2019, 8:33am UTC](https://forum.qiime2.org/t/error-whilst-using-v-search-join-pairs/12667/7 "2019-12-02T08:33:56Z")
HI @lghuggins, I certainly get errors in both vsearch in QIIME and vsearch alone when I try to work with reads that aren't paired, but maybe you're luckier than i am. If you can't get them to pair, my suggestion going forward would be to use single-end reads. Best, Justine ---
**Author:** ![lghuggins](https://forum.qiime2.org/letter_avatar_proxy/v4/letter/l/ba8739/32.png) [@lghuggins](https://forum.qiime2.org/u/lghuggins)\ **Post date:** [December 3, 2019, 11:53pm UTC](https://forum.qiime2.org/t/error-whilst-using-v-search-join-pairs/12667/9 "2019-12-03T23:53:50Z")
Just to confirm,what is the principal command from which to do --p-truncqual command? V-search? Cheers Lucas ---
**Author:** ![Nicholas\_Bokulich](https://forum.qiime2.org/user_avatar/forum.qiime2.org/nicholas_bokulich/32/19937_2.png) [@Nicholas\_Bokulich](https://forum.qiime2.org/u/Nicholas_Bokulich)\ **Post date:** [December 5, 2019, 4:35pm UTC](https://forum.qiime2.org/t/error-whilst-using-v-search-join-pairs/12667/10 "2019-12-05T16:35:54Z")
> [@lghuggins](#): > > what is the principal command from which to do --p-truncqual command? this is a parameter used by dada2 ---
**Author:** ![thermokarst](https://forum.qiime2.org/letter_avatar_proxy/v4/letter/t/8e7dd6/32.png) [@thermokarst](https://forum.qiime2.org/u/thermokarst)\ **Post date:** [November 9, 2020, 10:50pm UTC](https://forum.qiime2.org/t/error-whilst-using-v-search-join-pairs/12667/11 "2020-11-09T22:50:33Z")
A post was split to a new topic: [Looking for help joining paired-end reads](https://forum.qiime2.org/t/looking-for-help-joining-paired-end-reads/17394) ---
**Author:** ![thermokarst](https://forum.qiime2.org/letter_avatar_proxy/v4/letter/t/8e7dd6/32.png) [@thermokarst](https://forum.qiime2.org/u/thermokarst)\ **Post date:** [November 9, 2020, 10:50pm UTC](https://forum.qiime2.org/t/error-whilst-using-v-search-join-pairs/12667/12 "2020-11-09T22:50:41Z")