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 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
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 “</home/lucas/miniconda3/envs/qiime2-2019.7/lib/python3.6/site-packages/decorator.py:decorator-gen-131>”, 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.