join-pairs: plugin error from vsearch

Technical Support
1

Hello, First and foremost, thanks for providing such an outstanding piece of software.

I am in the process of making OTUs. I successfully imported the sequences as an artifact and ran cutadapt to remove the non biological sequences on the reads. I am getting hung up at the merge step. Vsearch plugin keeps throwing the same error.
I tried removing the sequence the initial error, but got the same error again with the next sequence down the line, so i dont think this is the same problem as another post about vsearch on the forum.

I've also tried running with a different version of Qiime, I've tried specifying --p-minmergelen.
Please let me know if you have any clues. All error details below. Let me know if you need additional information.

Thanks,
Gerid

Qiime2-2020.6

  • What is the exact command or commands you ran? Copy and paste please.
    qiime vsearch join-pairs --i-demultiplexed-seqs demux_trimmed.qza --o-joined-sequences demux-joined.qza
**Error message:**
**Plugin error from vsearch:**

**Command '['vsearch', '--fastq_mergepairs', '/tmp/qiime2-archive-5qdqdww3/98259609-1e49-4951-bec0-0639d21dad72/data/SMP-A-4-13_S2_92_L001_R1_001.fastq.gz', '--reverse', '/tmp/qiime2-archive-5qdqdww3/98259609-1e49-4951-bec0-0639d21dad72/data/SMP-A-4-13_S2_93_L001_R2_001.fastq.gz', '--fastqout', '/tmp/q2-SingleLanePerSampleSingleEndFastqDirFmt-kjcv9cl4/SMP-A-4-13_S2_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', '--minseqlength', '1', '--fasta_width', '0', '--threads', '1']' returned non-zero exit status 1.**

**Debug info has been saved to /tmp/qiime2-q2cli-err-cc4qfnm2.log**

> more /tmp/qiime2-q2cli-err-cc4qfnm2.log

/usr/local/conda/envs/qiime2-2020.6/lib/python3.6/site-packages/q2_vsearch/_join_pairs.py:93: YAMLLoadWarning: calling yaml.load() without Loader=... is deprecated, as the default Loader is unsafe. Please read https://msg.pyyaml.org/loa
d 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 /tmp/qiime2-archive-5qdqdww3/98259609-1e49-4951-bec0-0639d21dad72/data/SMP-A-4-13_S2_92_L001_R1_001.fastq.gz --reverse /tmp/qiime2-archive-5qdqdww3/98259609-1e49-4951-bec0-0639d21dad72/data/SMP-A-4-13
_S2_93_L001_R2_001.fastq.gz --fastqout /tmp/q2-SingleLanePerSampleSingleEndFastqDirFmt-kjcv9cl4/SMP-A-4-13_S2_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 --minseqlength 1 --fasta_width 0 --threads 1

Fatal error: Invalid options to command fastq_mergepairs
Invalid option(s): --minseqlength --fastq_qminout
The valid options for the fastq_mergepairs command are: --bzip2_decompress --eeout --eetabbedout --fasta_width --fastaout --fastaout_notmerged_fwd --fastaout_notmerged_rev --fastq_allowmergestagger --fastq_ascii --fastq_eeout --fastq_ma
xdiffpct --fastq_maxdiffs --fastq_maxee --fastq_maxlen --fastq_maxmergelen --fastq_maxns --fastq_minlen --fastq_minmergelen --fastq_minovlen --fastq_nostagger --fastq_qmax --fastq_qmaxout --fastq_qmin --fastq_truncqual --fastqout --fast
qout_notmerged_fwd --fastqout_notmerged_rev --gzip_decompress --label_suffix --log --no_progress --quiet --relabel --relabel_keep --relabel_md5 --relabel_sha1 --reverse --threads --xee --xsize
Traceback (most recent call last):
  File "/usr/local/conda/envs/qiime2-2020.6/lib/python3.6/site-packages/q2cli/commands.py", line 329, in __call__
    results = action(**arguments)
  File "<decorator-gen-131>", line 2, in join_pairs
  File "/usr/local/conda/envs/qiime2-2020.6/lib/python3.6/site-packages/qiime2/sdk/action.py", line 245, in bound_callable
    output_types, provenance)
  File "/usr/local/conda/envs/qiime2-2020.6/lib/python3.6/site-packages/qiime2/sdk/action.py", line 390, in _callable_executor_
    output_views = self._callable(**view_args)
  File "/usr/local/conda/envs/qiime2-2020.6/lib/python3.6/site-packages/q2_vsearch/_join_pairs.py", line 59, in join_pairs
    qmax, qmaxout, threads)
  File "/usr/local/conda/envs/qiime2-2020.6/lib/python3.6/site-packages/q2_vsearch/_join_pairs.py", line 146, in _join_pairs_w_command_output
    run_command(cmd)
  File "/usr/local/conda/envs/qiime2-2020.6/lib/python3.6/site-packages/q2_vsearch/_cluster_features.py", line 33, in run_command
    subprocess.run(cmd, check=True)
  File "/usr/local/conda/envs/qiime2-2020.6/lib/python3.6/subprocess.py", line 438, in run
    output=stdout, stderr=stderr)
subprocess.CalledProcessError: Command '['vsearch', '--fastq_mergepairs', '/tmp/qiime2-archive-5qdqdww3/98259609-1e49-4951-bec0-0639d21dad72/data/SMP-A-4-13_S2_92_L001_R1_001.fastq.gz', '--reverse', '/tmp/qiime2-archive-5qdqdww3/9825960
9-1e49-4951-bec0-0639d21dad72/data/SMP-A-4-13_S2_93_L001_R2_001.fastq.gz', '--fastqout', '/tmp/q2-SingleLanePerSampleSingleEndFastqDirFmt-kjcv9cl4/SMP-A-4-13_S2_0_L001_R1_001.fastq', '--fastq_ascii', '33', '--fastq_minlen', '1', '--fast
q_minovlen', '10', '--fastq_maxdiffs', '10', '--fastq_qmin', '0', '--fastq_qminout', '0', '--fastq_qmax', '41', '--fastq_qmaxout', '41', '--minseqlength', '1', '--fasta_width', '0', '--threads', '1']' returned non-zero exit status 1.
2

Hi @gollison

Just the first thing I noticed, vsearch is trying to merge
forward read from sample 'SMP-A-4-13_S2_92' and reverse read from sample 'SMP-A-4-13_S2_93', is this the correct pairing?
How did you import the data?

hope it helps

1 Like
3

Good eye. This is strange indeed. Those numbers are not in my manifest file or the fastq name itself. It must have been incoorporated during the import process and describes the samples in a sequence?

Anyway, I imported like this:

> qiime tools import --type 'SampleData[PairedEndSequencesWithQuality]' --input-path trimmed_manifest.txt --output-path demux.qza --input-format PairedEndFastqManifestPhred33&

my manifest looks like this:

SMPier-Day10-A-04-25-2018_S10,$PWD/trimmed_seq_dir/SMPier-Day10-A-04-25-2018_S10_R1_PE.fastq.gz,forward

SMPier-Day10-A-04-25-2018_S10,$PWD/trimmed_seq_dir/SMPier-Day10-A-04-25-2018_S10_R2_PE.fastq.gz,reverse

SMPier-Day10-B-04-25-2018_S25,$PWD/trimmed_seq_dir/SMPier-Day10-B-04-25-2018_S25_R1_PE.fastq.gz,forward

SMPier-Day10-B-04-25-2018_S25,$PWD/trimmed_seq_dir/SMPier-Day10-B-04-25-2018_S25_R2_PE.fastq.gz,reverse

SMPier-Day10-C-04-25-2018_S40,$PWD/trimmed_seq_dir/SMPier-Day10-C-04-25-2018_S40_R1_PE.fastq.gz,forward

SMPier-Day10-C-04-25-2018_S40,$PWD/trimmed_seq_dir/SMPier-Day10-C-04-25-2018_S40_R2_PE.fastq.gz,reverse

SMPier-Day11-A-04-26-2018_S11,$PWD/trimmed_seq_dir/SMPier-Day11-A-04-26-2018_S11_R1_PE.fastq.gz,forward

SMPier-Day11-A-04-26-2018_S11,$PWD/trimmed_seq_dir/SMPier-Day11-A-04-26-2018_S11_R2_PE.fastq.gz,reverse

SMPier-Day11-B-04-26-2018_S26,$PWD/trimmed_seq_dir/SMPier-Day11-B-04-26-2018_S26_R1_PE.fastq.gz,forward

SMPier-Day11-B-04-26-2018_S26,$PWD/trimmed_seq_dir/SMPier-Day11-B-04-26-2018_S26_R2_PE.fastq.gz,reverse

SMPier-Day11-C-04-26-2018_S41,$PWD/trimmed_seq_dir/SMPier-Day11-C-04-26-2018_S41_R1_PE.fastq.gz,forward

SMPier-Day11-C-04-26-2018_S41,$PWD/trimmed_seq_dir/SMPier-Day11-C-04-26-2018_S41_R2_PE.fastq.gz,reverse

SMPier-Day12-A-04-27-2018_S12,$PWD/trimmed_seq_dir/SMPier-Day12-A-04-27-2018_S12_R1_PE.fastq.gz,forward

SMPier-Day12-A-04-27-2018_S12,$PWD/trimmed_seq_dir/SMPier-Day12-A-04-27-2018_S12_R2_PE.fastq.gz,reverse

SMPier-Day12-B-04-27-2018_S27,$PWD/trimmed_seq_dir/SMPier-Day12-B-04-27-2018_S27_R1_PE.fastq.gz,forward

SMPier-Day12-B-04-27-2018_S27,$PWD/trimmed_seq_dir/SMPier-Day12-B-04-27-2018_S27_R2_PE.fastq.gz,reverse

SMPier-Day12-C-04-27-2018_S42,$PWD/trimmed_seq_dir/SMPier-Day12-C-04-27-2018_S42_R1_PE.fastq.gz,forward

SMPier-Day12-C-04-27-2018_S42,$PWD/trimmed_seq_dir/SMPier-Day12-C-04-27-2018_S42_R2_PE.fastq.gz,reverse

SMPier-Day13-A-04-28-2018_S13,$PWD/trimmed_seq_dir/SMPier-Day13-A-04-28-2018_S13_R1_PE.fastq.gz,forward

SMPier-Day13-A-04-28-2018_S13,$PWD/trimmed_seq_dir/SMPier-Day13-A-04-28-2018_S13_R2_PE.fastq.gz,reverse

SMPier-Day13-B-04-28-2018_S28,$PWD/trimmed_seq_dir/SMPier-Day13-B-04-28-2018_S28_R1_PE.fastq.gz,forward

SMPier-Day13-B-04-28-2018_S28,$PWD/trimmed_seq_dir/SMPier-Day13-B-04-28-2018_S28_R2_PE.fastq.gz,reverse

SMPier-Day13-C-04-28-2018_S43,$PWD/trimmed_seq_dir/SMPier-Day13-C-04-28-2018_S43_R1_PE.fastq.gz,forward

SMPier-Day13-C-04-28-2018_S43,$PWD/trimmed_seq_dir/SMPier-Day13-C-04-28-2018_S43_R2_PE.fastq.gz,reverse

SMPier-Day14-A-04-29-2018_S14,$PWD/trimmed_seq_dir/SMPier-Day14-A-04-29-2018_S14_R1_PE.fastq.gz,forward

SMPier-Day14-A-04-29-2018_S14,$PWD/trimmed_seq_dir/SMPier-Day14-A-04-29-2018_S14_R2_PE.fastq.gz,reverse

SMPier-Day14-B-04-29-2018_S29,$PWD/trimmed_seq_dir/SMPier-Day14-B-04-29-2018_S29_R1_PE.fastq.gz,forward

SMPier-Day14-B-04-29-2018_S29,$PWD/trimmed_seq_dir/SMPier-Day14-B-04-29-2018_S29_R2_PE.fastq.gz,reverse

SMPier-Day14-C-04-29-2018_S44,$PWD/trimmed_seq_dir/SMPier-Day14-C-04-29-2018_S44_R1_PE.fastq.gz,forward

SMPier-Day14-C-04-29-2018_S44,$PWD/trimmed_seq_dir/SMPier-Day14-C-04-29-2018_S44_R2_PE.fastq.gz,reverse

SMPier-Day15-A-04-30-2018_S15,$PWD/trimmed_seq_dir/SMPier-Day15-A-04-30-2018_S15_R1_PE.fastq.gz,forward

SMPier-Day15-A-04-30-2018_S15,$PWD/trimmed_seq_dir/SMPier-Day15-A-04-30-2018_S15_R2_PE.fastq.gz,reverse

SMPier-Day15-B-04-30-2018_S30,$PWD/trimmed_seq_dir/SMPier-Day15-B-04-30-2018_S30_R1_PE.fastq.gz,forward

SMPier-Day15-B-04-30-2018_S30,$PWD/trimmed_seq_dir/SMPier-Day15-B-04-30-2018_S30_R2_PE.fastq.gz,reverse

SMPier-Day15-C-04-30-2018_S45,$PWD/trimmed_seq_dir/SMPier-Day15-C-04-30-2018_S45_R1_PE.fastq.gz,forward

SMPier-Day15-C-04-30-2018_S45,$PWD/trimmed_seq_dir/SMPier-Day15-C-04-30-2018_S45_R2_PE.fastq.gz,reverse

SMPier-Day1-A-04-16-2018_S1,$PWD/trimmed_seq_dir/SMPier-Day1-A-04-16-2018_S1_R1_PE.fastq.gz,forward

SMPier-Day1-A-04-16-2018_S1,$PWD/trimmed_seq_dir/SMPier-Day1-A-04-16-2018_S1_R2_PE.fastq.gz,reverse

SMPier-Day1-B-04-16-2018_S16,$PWD/trimmed_seq_dir/SMPier-Day1-B-04-16-2018_S16_R1_PE.fastq.gz,forward

SMPier-Day1-B-04-16-2018_S16,$PWD/trimmed_seq_dir/SMPier-Day1-B-04-16-2018_S16_R2_PE.fastq.gz,reverse

SMPier-Day1-C-04-16-2018_S31,$PWD/trimmed_seq_dir/SMPier-Day1-C-04-16-2018_S31_R1_PE.fastq.gz,forward

SMPier-Day1-C-04-16-2018_S31,$PWD/trimmed_seq_dir/SMPier-Day1-C-04-16-2018_S31_R2_PE.fastq.gz,reverse

SMPier-Day2-A-04-17-2018_S2,$PWD/trimmed_seq_dir/SMPier-Day2-A-04-17-2018_S2_R1_PE.fastq.gz,forward

SMPier-Day2-A-04-17-2018_S2,$PWD/trimmed_seq_dir/SMPier-Day2-A-04-17-2018_S2_R2_PE.fastq.gz,reverse

SMPier-Day2-B-04-17-2018_S17,$PWD/trimmed_seq_dir/SMPier-Day2-B-04-17-2018_S17_R1_PE.fastq.gz,forward

SMPier-Day2-B-04-17-2018_S17,$PWD/trimmed_seq_dir/SMPier-Day2-B-04-17-2018_S17_R2_PE.fastq.gz,reverse

SMPier-Day2-C-04-17-2018_S32,$PWD/trimmed_seq_dir/SMPier-Day2-C-04-17-2018_S32_R1_PE.fastq.gz,forward

SMPier-Day2-C-04-17-2018_S32,$PWD/trimmed_seq_dir/SMPier-Day2-C-04-17-2018_S32_R2_PE.fastq.gz,reverse

SMPier-Day3-A-04-18-2018_S3,$PWD/trimmed_seq_dir/SMPier-Day3-A-04-18-2018_S3_R1_PE.fastq.gz,forward

SMPier-Day3-A-04-18-2018_S3,$PWD/trimmed_seq_dir/SMPier-Day3-A-04-18-2018_S3_R2_PE.fastq.gz,reverse

SMPier-Day3-B-04-18-2018_S18,$PWD/trimmed_seq_dir/SMPier-Day3-B-04-18-2018_S18_R1_PE.fastq.gz,forward

SMPier-Day3-B-04-18-2018_S18,$PWD/trimmed_seq_dir/SMPier-Day3-B-04-18-2018_S18_R2_PE.fastq.gz,reverse

SMPier-Day3-C-04-18-2018_S33,$PWD/trimmed_seq_dir/SMPier-Day3-C-04-18-2018_S33_R1_PE.fastq.gz,forward

SMPier-Day3-C-04-18-2018_S33,$PWD/trimmed_seq_dir/SMPier-Day3-C-04-18-2018_S33_R2_PE.fastq.gz,reverse

SMPier-Day4-A-04-19-2018_S4,$PWD/trimmed_seq_dir/SMPier-Day4-A-04-19-2018_S4_R1_PE.fastq.gz,forward

SMPier-Day4-A-04-19-2018_S4,$PWD/trimmed_seq_dir/SMPier-Day4-A-04-19-2018_S4_R2_PE.fastq.gz,reverse

SMPier-Day4-B-04-19-2018_S19,$PWD/trimmed_seq_dir/SMPier-Day4-B-04-19-2018_S19_R1_PE.fastq.gz,forward

SMPier-Day4-B-04-19-2018_S19,$PWD/trimmed_seq_dir/SMPier-Day4-B-04-19-2018_S19_R2_PE.fastq.gz,reverse

SMPier-Day4-C-04-19-2018_S34,$PWD/trimmed_seq_dir/SMPier-Day4-C-04-19-2018_S34_R1_PE.fastq.gz,forward

SMPier-Day4-C-04-19-2018_S34,$PWD/trimmed_seq_dir/SMPier-Day4-C-04-19-2018_S34_R2_PE.fastq.gz,reverse

SMPier-Day5-A-04-20-2018_S5,$PWD/trimmed_seq_dir/SMPier-Day5-A-04-20-2018_S5_R1_PE.fastq.gz,forward

SMPier-Day5-A-04-20-2018_S5,$PWD/trimmed_seq_dir/SMPier-Day5-A-04-20-2018_S5_R2_PE.fastq.gz,reverse

SMPier-Day5-B-04-20-2018_S20,$PWD/trimmed_seq_dir/SMPier-Day5-B-04-20-2018_S20_R1_PE.fastq.gz,forward

SMPier-Day5-B-04-20-2018_S20,$PWD/trimmed_seq_dir/SMPier-Day5-B-04-20-2018_S20_R2_PE.fastq.gz,reverse

SMPier-Day5-C-04-20-2018_S35,$PWD/trimmed_seq_dir/SMPier-Day5-C-04-20-2018_S35_R1_PE.fastq.gz,forward

SMPier-Day5-C-04-20-2018_S35,$PWD/trimmed_seq_dir/SMPier-Day5-C-04-20-2018_S35_R2_PE.fastq.gz,reverse

SMPier-Day6-A-04-21-2018_S6,$PWD/trimmed_seq_dir/SMPier-Day6-A-04-21-2018_S6_R1_PE.fastq.gz,forward

SMPier-Day6-A-04-21-2018_S6,$PWD/trimmed_seq_dir/SMPier-Day6-A-04-21-2018_S6_R2_PE.fastq.gz,reverse

SMPier-Day6-B-04-21-2018_S21,$PWD/trimmed_seq_dir/SMPier-Day6-B-04-21-2018_S21_R1_PE.fastq.gz,forward

SMPier-Day6-B-04-21-2018_S21,$PWD/trimmed_seq_dir/SMPier-Day6-B-04-21-2018_S21_R2_PE.fastq.gz,reverse

SMPier-Day6-C-04-21-2018_S36,$PWD/trimmed_seq_dir/SMPier-Day6-C-04-21-2018_S36_R1_PE.fastq.gz,forward

SMPier-Day6-C-04-21-2018_S36,$PWD/trimmed_seq_dir/SMPier-Day6-C-04-21-2018_S36_R2_PE.fastq.gz,reverse

SMPier-Day7-A-04-22-2018_S7,$PWD/trimmed_seq_dir/SMPier-Day7-A-04-22-2018_S7_R1_PE.fastq.gz,forward

SMPier-Day7-A-04-22-2018_S7,$PWD/trimmed_seq_dir/SMPier-Day7-A-04-22-2018_S7_R2_PE.fastq.gz,reverse

SMPier-Day7-B-04-22-2018_S22,$PWD/trimmed_seq_dir/SMPier-Day7-B-04-22-2018_S22_R1_PE.fastq.gz,forward

SMPier-Day7-B-04-22-2018_S22,$PWD/trimmed_seq_dir/SMPier-Day7-B-04-22-2018_S22_R2_PE.fastq.gz,reverse

SMPier-Day7-C-04-22-2018_S37,$PWD/trimmed_seq_dir/SMPier-Day7-C-04-22-2018_S37_R1_PE.fastq.gz,forward

SMPier-Day7-C-04-22-2018_S37,$PWD/trimmed_seq_dir/SMPier-Day7-C-04-22-2018_S37_R2_PE.fastq.gz,reverse

SMPier-Day8-A-04-23-2018_S8,$PWD/trimmed_seq_dir/SMPier-Day8-A-04-23-2018_S8_R1_PE.fastq.gz,forward

SMPier-Day8-A-04-23-2018_S8,$PWD/trimmed_seq_dir/SMPier-Day8-A-04-23-2018_S8_R2_PE.fastq.gz,reverse

SMPier-Day8-B-04-23-2018_S23,$PWD/trimmed_seq_dir/SMPier-Day8-B-04-23-2018_S23_R1_PE.fastq.gz,forward

SMPier-Day8-B-04-23-2018_S23,$PWD/trimmed_seq_dir/SMPier-Day8-B-04-23-2018_S23_R2_PE.fastq.gz,reverse

SMPier-Day8-C-04-23-2018_S38,$PWD/trimmed_seq_dir/SMPier-Day8-C-04-23-2018_S38_R1_PE.fastq.gz,forward

SMPier-Day8-C-04-23-2018_S38,$PWD/trimmed_seq_dir/SMPier-Day8-C-04-23-2018_S38_R2_PE.fastq.gz,reverse

SMPier-Day9-A-04-24-2018_S9,$PWD/trimmed_seq_dir/SMPier-Day9-A-04-24-2018_S9_R1_PE.fastq.gz,forward

SMPier-Day9-A-04-24-2018_S9,$PWD/trimmed_seq_dir/SMPier-Day9-A-04-24-2018_S9_R2_PE.fastq.gz,reverse

SMPier-Day9-B-04-24-2018_S24,$PWD/trimmed_seq_dir/SMPier-Day9-B-04-24-2018_S24_R1_PE.fastq.gz,forward

SMPier-Day9-B-04-24-2018_S24,$PWD/trimmed_seq_dir/SMPier-Day9-B-04-24-2018_S24_R2_PE.fastq.gz,reverse

SMPier-Day9-C-04-24-2018_S39,$PWD/trimmed_seq_dir/SMPier-Day9-C-04-24-2018_S39_R1_PE.fastq.gz,forward

SMPier-Day9-C-04-24-2018_S39,$PWD/trimmed_seq_dir/SMPier-Day9-C-04-24-2018_S39_R2_PE.fastq.gz,reverse

blank-H20_S34,$PWD/trimmed_seq_dir/blank-H20_S34_R1_PE.fastq.gz,forward

blank-H20_S34,$PWD/trimmed_seq_dir/blank-H20_S34_R2_PE.fastq.gz,reverse

SMP-A-4-13_S2,$PWD/trimmed_seq_dir/SMP-A-4-13_S2_R1_PE.fastq.gz,forward

SMP-A-4-13_S2,$PWD/trimmed_seq_dir/SMP-A-4-13_S2_R2_PE.fastq.gz,reverse

SMP-A-4-15_S3,$PWD/trimmed_seq_dir/SMP-A-4-15_S3_R1_PE.fastq.gz,forward

SMP-A-4-15_S3,$PWD/trimmed_seq_dir/SMP-A-4-15_S3_R2_PE.fastq.gz,reverse

SMP-A-4-16_S4,$PWD/trimmed_seq_dir/SMP-A-4-16_S4_R1_PE.fastq.gz,forward

SMP-A-4-16_S4,$PWD/trimmed_seq_dir/SMP-A-4-16_S4_R2_PE.fastq.gz,reverse

SMP-A-4-17_S5,$PWD/trimmed_seq_dir/SMP-A-4-17_S5_R1_PE.fastq.gz,forward

SMP-A-4-17_S5,$PWD/trimmed_seq_dir/SMP-A-4-17_S5_R2_PE.fastq.gz,reverse

SMP-A-4-18_S6,$PWD/trimmed_seq_dir/SMP-A-4-18_S6_R1_PE.fastq.gz,forward

SMP-A-4-18_S6,$PWD/trimmed_seq_dir/SMP-A-4-18_S6_R2_PE.fastq.gz,reverse

SMP-A-4-19_S7,$PWD/trimmed_seq_dir/SMP-A-4-19_S7_R1_PE.fastq.gz,forward

SMP-A-4-19_S7,$PWD/trimmed_seq_dir/SMP-A-4-19_S7_R2_PE.fastq.gz,reverse

SMP-A-4-20_S8,$PWD/trimmed_seq_dir/SMP-A-4-20_S8_R1_PE.fastq.gz,forward

SMP-A-4-20_S8,$PWD/trimmed_seq_dir/SMP-A-4-20_S8_R2_PE.fastq.gz,reverse

SMP-A-4-21_S9,$PWD/trimmed_seq_dir/SMP-A-4-21_S9_R1_PE.fastq.gz,forward

SMP-A-4-21_S9,$PWD/trimmed_seq_dir/SMP-A-4-21_S9_R2_PE.fastq.gz,reverse

SMP-A-4-22_S10,$PWD/trimmed_seq_dir/SMP-A-4-22_S10_R1_PE.fastq.gz,forward

SMP-A-4-22_S10,$PWD/trimmed_seq_dir/SMP-A-4-22_S10_R2_PE.fastq.gz,reverse

SMP-A-4-23_S11,$PWD/trimmed_seq_dir/SMP-A-4-23_S11_R1_PE.fastq.gz,forward

SMP-A-4-23_S11,$PWD/trimmed_seq_dir/SMP-A-4-23_S11_R2_PE.fastq.gz,reverse

SMP-A-4-24_S12,$PWD/trimmed_seq_dir/SMP-A-4-24_S12_R1_PE.fastq.gz,forward

SMP-A-4-24_S12,$PWD/trimmed_seq_dir/SMP-A-4-24_S12_R2_PE.fastq.gz,reverse

SMP-A-4-25_S13,$PWD/trimmed_seq_dir/SMP-A-4-25_S13_R1_PE.fastq.gz,forward

SMP-A-4-25_S13,$PWD/trimmed_seq_dir/SMP-A-4-25_S13_R2_PE.fastq.gz,reverse

SMP-A-4-26_S14,$PWD/trimmed_seq_dir/SMP-A-4-26_S14_R1_PE.fastq.gz,forward

SMP-A-4-26_S14,$PWD/trimmed_seq_dir/SMP-A-4-26_S14_R2_PE.fastq.gz,reverse

SMP-A-4-27_S15,$PWD/trimmed_seq_dir/SMP-A-4-27_S15_R1_PE.fastq.gz,forward

SMP-A-4-27_S15,$PWD/trimmed_seq_dir/SMP-A-4-27_S15_R2_PE.fastq.gz,reverse

SMP-A-4-28_S16,$PWD/trimmed_seq_dir/SMP-A-4-28_S16_R1_PE.fastq.gz,forward

SMP-A-4-28_S16,$PWD/trimmed_seq_dir/SMP-A-4-28_S16_R2_PE.fastq.gz,reverse

SMP-A-4-29_S17,$PWD/trimmed_seq_dir/SMP-A-4-29_S17_R1_PE.fastq.gz,forward

SMP-A-4-29_S17,$PWD/trimmed_seq_dir/SMP-A-4-29_S17_R2_PE.fastq.gz,reverse

SMP-A-4-30_S18,$PWD/trimmed_seq_dir/SMP-A-4-30_S18_R1_PE.fastq.gz,forward

SMP-A-4-30_S18,$PWD/trimmed_seq_dir/SMP-A-4-30_S18_R2_PE.fastq.gz,reverse

SMP-A-5-01_S19,$PWD/trimmed_seq_dir/SMP-A-5-01_S19_R1_PE.fastq.gz,forward

SMP-A-5-01_S19,$PWD/trimmed_seq_dir/SMP-A-5-01_S19_R2_PE.fastq.gz,reverse

SMP-A-5-02_S20,$PWD/trimmed_seq_dir/SMP-A-5-02_S20_R1_PE.fastq.gz,forward

SMP-A-5-02_S20,$PWD/trimmed_seq_dir/SMP-A-5-02_S20_R2_PE.fastq.gz,reverse

SMP-A-5-03_S21,$PWD/trimmed_seq_dir/SMP-A-5-03_S21_R1_PE.fastq.gz,forward

SMP-A-5-03_S21,$PWD/trimmed_seq_dir/SMP-A-5-03_S21_R2_PE.fastq.gz,reverse

SMP-A-5-04_S22,$PWD/trimmed_seq_dir/SMP-A-5-04_S22_R1_PE.fastq.gz,forward

SMP-A-5-04_S22,$PWD/trimmed_seq_dir/SMP-A-5-04_S22_R2_PE.fastq.gz,reverse

SMP-A-5-05_S23,$PWD/trimmed_seq_dir/SMP-A-5-05_S23_R1_PE.fastq.gz,forward

SMP-A-5-05_S23,$PWD/trimmed_seq_dir/SMP-A-5-05_S23_R2_PE.fastq.gz,reverse

SMP-B-4-13_S25,$PWD/trimmed_seq_dir/SMP-B-4-13_S25_R1_PE.fastq.gz,forward

SMP-B-4-13_S25,$PWD/trimmed_seq_dir/SMP-B-4-13_S25_R2_PE.fastq.gz,reverse

SMP-B-4-15_S26,$PWD/trimmed_seq_dir/SMP-B-4-15_S26_R1_PE.fastq.gz,forward

SMP-B-4-15_S26,$PWD/trimmed_seq_dir/SMP-B-4-15_S26_R2_PE.fastq.gz,reverse

SMP-B-4-16_S27,$PWD/trimmed_seq_dir/SMP-B-4-16_S27_R1_PE.fastq.gz,forward

SMP-B-4-16_S27,$PWD/trimmed_seq_dir/SMP-B-4-16_S27_R2_PE.fastq.gz,reverse

SMP-B-4-17_S28,$PWD/trimmed_seq_dir/SMP-B-4-17_S28_R1_PE.fastq.gz,forward

SMP-B-4-17_S28,$PWD/trimmed_seq_dir/SMP-B-4-17_S28_R2_PE.fastq.gz,reverse

SMP-B-4-18_S29,$PWD/trimmed_seq_dir/SMP-B-4-18_S29_R1_PE.fastq.gz,forward

SMP-B-4-18_S29,$PWD/trimmed_seq_dir/SMP-B-4-18_S29_R2_PE.fastq.gz,reverse

SMP-B-4-19_S30,$PWD/trimmed_seq_dir/SMP-B-4-19_S30_R1_PE.fastq.gz,forward

SMP-B-4-19_S30,$PWD/trimmed_seq_dir/SMP-B-4-19_S30_R2_PE.fastq.gz,reverse

SMP-B-4-20_S31,$PWD/trimmed_seq_dir/SMP-B-4-20_S31_R1_PE.fastq.gz,forward

SMP-B-4-20_S31,$PWD/trimmed_seq_dir/SMP-B-4-20_S31_R2_PE.fastq.gz,reverse

SMP-B-4-21_S32,$PWD/trimmed_seq_dir/SMP-B-4-21_S32_R1_PE.fastq.gz,forward

SMP-B-4-21_S32,$PWD/trimmed_seq_dir/SMP-B-4-21_S32_R2_PE.fastq.gz,reverse

SMP-B-4-22_S33,$PWD/trimmed_seq_dir/SMP-B-4-22_S33_R1_PE.fastq.gz,forward

SMP-B-4-22_S33,$PWD/trimmed_seq_dir/SMP-B-4-22_S33_R2_PE.fastq.gz,reverse

SMP-B-4-23_S34,$PWD/trimmed_seq_dir/SMP-B-4-23_S34_R1_PE.fastq.gz,forward

SMP-B-4-23_S34,$PWD/trimmed_seq_dir/SMP-B-4-23_S34_R2_PE.fastq.gz,reverse

SMP-B-4-24_S35,$PWD/trimmed_seq_dir/SMP-B-4-24_S35_R1_PE.fastq.gz,forward

SMP-B-4-24_S35,$PWD/trimmed_seq_dir/SMP-B-4-24_S35_R2_PE.fastq.gz,reverse

SMP-B-4-25_S1,$PWD/trimmed_seq_dir/SMP-B-4-25_S1_R1_PE.fastq.gz,forward

SMP-B-4-25_S1,$PWD/trimmed_seq_dir/SMP-B-4-25_S1_R2_PE.fastq.gz,reverse

SMP-B-4-26_S2,$PWD/trimmed_seq_dir/SMP-B-4-26_S2_R1_PE.fastq.gz,forward

SMP-B-4-26_S2,$PWD/trimmed_seq_dir/SMP-B-4-26_S2_R2_PE.fastq.gz,reverse

SMP-B-4-27_S3,$PWD/trimmed_seq_dir/SMP-B-4-27_S3_R1_PE.fastq.gz,forward

SMP-B-4-27_S3,$PWD/trimmed_seq_dir/SMP-B-4-27_S3_R2_PE.fastq.gz,reverse

SMP-B-4-28_S4,$PWD/trimmed_seq_dir/SMP-B-4-28_S4_R1_PE.fastq.gz,forward

SMP-B-4-28_S4,$PWD/trimmed_seq_dir/SMP-B-4-28_S4_R2_PE.fastq.gz,reverse

SMP-B-4-29_S5,$PWD/trimmed_seq_dir/SMP-B-4-29_S5_R1_PE.fastq.gz,forward

SMP-B-4-29_S5,$PWD/trimmed_seq_dir/SMP-B-4-29_S5_R2_PE.fastq.gz,reverse

SMP-B-4-30_S6,$PWD/trimmed_seq_dir/SMP-B-4-30_S6_R1_PE.fastq.gz,forward

SMP-B-4-30_S6,$PWD/trimmed_seq_dir/SMP-B-4-30_S6_R2_PE.fastq.gz,reverse

SMP-B-5-01_S7,$PWD/trimmed_seq_dir/SMP-B-5-01_S7_R1_PE.fastq.gz,forward

SMP-B-5-01_S7,$PWD/trimmed_seq_dir/SMP-B-5-01_S7_R2_PE.fastq.gz,reverse

SMP-B-5-02_S8,$PWD/trimmed_seq_dir/SMP-B-5-02_S8_R1_PE.fastq.gz,forward

SMP-B-5-02_S8,$PWD/trimmed_seq_dir/SMP-B-5-02_S8_R2_PE.fastq.gz,reverse

SMP-B-5-03_S9,$PWD/trimmed_seq_dir/SMP-B-5-03_S9_R1_PE.fastq.gz,forward

SMP-B-5-03_S9,$PWD/trimmed_seq_dir/SMP-B-5-03_S9_R2_PE.fastq.gz,reverse

SMP-B-5-04_S10,$PWD/trimmed_seq_dir/SMP-B-5-04_S10_R1_PE.fastq.gz,forward

SMP-B-5-04_S10,$PWD/trimmed_seq_dir/SMP-B-5-04_S10_R2_PE.fastq.gz,reverse

SMP-B-5-05_S11,$PWD/trimmed_seq_dir/SMP-B-5-05_S11_R1_PE.fastq.gz,forward

SMP-B-5-05_S11,$PWD/trimmed_seq_dir/SMP-B-5-05_S11_R2_PE.fastq.gz,reverse

SMP-C-4-13_S13,$PWD/trimmed_seq_dir/SMP-C-4-13_S13_R1_PE.fastq.gz,forward

SMP-C-4-13_S13,$PWD/trimmed_seq_dir/SMP-C-4-13_S13_R2_PE.fastq.gz,reverse

SMP-C-4-15_S14,$PWD/trimmed_seq_dir/SMP-C-4-15_S14_R1_PE.fastq.gz,forward

SMP-C-4-15_S14,$PWD/trimmed_seq_dir/SMP-C-4-15_S14_R2_PE.fastq.gz,reverse

SMP-C-4-16_S15,$PWD/trimmed_seq_dir/SMP-C-4-16_S15_R1_PE.fastq.gz,forward

SMP-C-4-16_S15,$PWD/trimmed_seq_dir/SMP-C-4-16_S15_R2_PE.fastq.gz,reverse

SMP-C-4-17_S16,$PWD/trimmed_seq_dir/SMP-C-4-17_S16_R1_PE.fastq.gz,forward

SMP-C-4-17_S16,$PWD/trimmed_seq_dir/SMP-C-4-17_S16_R2_PE.fastq.gz,reverse

SMP-C-4-18_S17,$PWD/trimmed_seq_dir/SMP-C-4-18_S17_R1_PE.fastq.gz,forward

SMP-C-4-18_S17,$PWD/trimmed_seq_dir/SMP-C-4-18_S17_R2_PE.fastq.gz,reverse

SMP-C-4-19_S18,$PWD/trimmed_seq_dir/SMP-C-4-19_S18_R1_PE.fastq.gz,forward

SMP-C-4-19_S18,$PWD/trimmed_seq_dir/SMP-C-4-19_S18_R2_PE.fastq.gz,reverse

SMP-C-4-20_S19,$PWD/trimmed_seq_dir/SMP-C-4-20_S19_R1_PE.fastq.gz,forward

SMP-C-4-20_S19,$PWD/trimmed_seq_dir/SMP-C-4-20_S19_R2_PE.fastq.gz,reverse

SMP-C-4-21_S20,$PWD/trimmed_seq_dir/SMP-C-4-21_S20_R1_PE.fastq.gz,forward

SMP-C-4-21_S20,$PWD/trimmed_seq_dir/SMP-C-4-21_S20_R2_PE.fastq.gz,reverse

SMP-C-4-22_S21,$PWD/trimmed_seq_dir/SMP-C-4-22_S21_R1_PE.fastq.gz,forward

SMP-C-4-22_S21,$PWD/trimmed_seq_dir/SMP-C-4-22_S21_R2_PE.fastq.gz,reverse

SMP-C-4-23_S22,$PWD/trimmed_seq_dir/SMP-C-4-23_S22_R1_PE.fastq.gz,forward

SMP-C-4-23_S22,$PWD/trimmed_seq_dir/SMP-C-4-23_S22_R2_PE.fastq.gz,reverse

SMP-C-4-24_S23,$PWD/trimmed_seq_dir/SMP-C-4-24_S23_R1_PE.fastq.gz,forward

SMP-C-4-24_S23,$PWD/trimmed_seq_dir/SMP-C-4-24_S23_R2_PE.fastq.gz,reverse

SMP-C-4-26_S25,$PWD/trimmed_seq_dir/SMP-C-4-26_S25_R1_PE.fastq.gz,forward

SMP-C-4-26_S25,$PWD/trimmed_seq_dir/SMP-C-4-26_S25_R2_PE.fastq.gz,reverse

SMP-C-4-27_S26,$PWD/trimmed_seq_dir/SMP-C-4-27_S26_R1_PE.fastq.gz,forward

SMP-C-4-27_S26,$PWD/trimmed_seq_dir/SMP-C-4-27_S26_R2_PE.fastq.gz,reverse

SMP-C-4-28_S27,$PWD/trimmed_seq_dir/SMP-C-4-28_S27_R1_PE.fastq.gz,forward

SMP-C-4-28_S27,$PWD/trimmed_seq_dir/SMP-C-4-28_S27_R2_PE.fastq.gz,reverse

SMP-C-4-29_S28,$PWD/trimmed_seq_dir/SMP-C-4-29_S28_R1_PE.fastq.gz,forward

SMP-C-4-29_S28,$PWD/trimmed_seq_dir/SMP-C-4-29_S28_R2_PE.fastq.gz,reverse

SMP-C-4-30_S29,$PWD/trimmed_seq_dir/SMP-C-4-30_S29_R1_PE.fastq.gz,forward

SMP-C-4-30_S29,$PWD/trimmed_seq_dir/SMP-C-4-30_S29_R2_PE.fastq.gz,reverse

SMP-C-5-01_S30,$PWD/trimmed_seq_dir/SMP-C-5-01_S30_R1_PE.fastq.gz,forward

SMP-C-5-01_S30,$PWD/trimmed_seq_dir/SMP-C-5-01_S30_R2_PE.fastq.gz,reverse

SMP-C-5-02_S31,$PWD/trimmed_seq_dir/SMP-C-5-02_S31_R1_PE.fastq.gz,forward

SMP-C-5-02_S31,$PWD/trimmed_seq_dir/SMP-C-5-02_S31_R2_PE.fastq.gz,reverse

SMP-C-5-03_S32,$PWD/trimmed_seq_dir/SMP-C-5-03_S32_R1_PE.fastq.gz,forward

SMP-C-5-03_S32,$PWD/trimmed_seq_dir/SMP-C-5-03_S32_R2_PE.fastq.gz,reverse

SMP-C-5-04_S33,$PWD/trimmed_seq_dir/SMP-C-5-04_S33_R1_PE.fastq.gz,forward

SMP-C-5-04_S33,$PWD/trimmed_seq_dir/SMP-C-5-04_S33_R2_PE.fastq.gz,reverse

SMP-C-5-05_S35,$PWD/trimmed_seq_dir/SMP-C-5-05_S35_R1_PE.fastq.gz,forward

SMP-C-5-05_S35,$PWD/trimmed_seq_dir/SMP-C-5-05_S35_R2_PE.fastq.gz,reverse
4

I had a look at the demux.qzv and found that those numbers (92 and 93) dont exist in the sample name there either so they must be getting incorporated during some temporary vsearch step.

5

The CASAVA barcode number is a red herring, I think. The bulk of the error message appears to be this:

This error message is coming from vsearch, it is complaining that several flags are "invalid options", which is a very strong indication that somehow you have a very old version of vsearch leaking into your QIIME 2 environment. QIIME 2020.6 shipped with vsearch 2.7.0. If you activate your QIIME 2 env and run vsearch --version and which vsearch, what do you see?

6

$ vsearch --version

vsearch v2.13.3_linux_x86_64, 251.6GB RAM, 128 cores

Rognes T, Flouri T, Nichols B, Quince C, Mahe F (2016)

VSEARCH: a versatile open source tool for metagenomics

PeerJ 4:e2584 doi: 10.7717/peerj.2584 VSEARCH: a versatile open source tool for metagenomics [PeerJ]

It appears to be different from 2.7.0 but newer? (v2.13.3).

7

Thanks @gollison!

Well that'll do it!

Yeah, either way, fundamentally incompatible with q2-vsearch.

Looks like you missed this part of my request above:

Please run that command and share the results.

Clearly something in your computation environment is not playing nicely with your QIIME 2 conda environment. It would also be helpful if you ran the command echo $PATH in your QIIME 2 conda env so that we can see what that looks like.

:qiime2:

8

$ which vsearch

/usr/local/bioinf/Mothur/mothur/vsearch

It appears it is using an externally installed vsearch?

9

One more request from above, @gollison:

10

$ echo $PATH

/usr/local/bioinf/FastQC:/usr/local/bioinf/spades/SPAdes-3.12.0-Linux/bin:/usr/local/bioinf/Mothur/mothur:/home/gerid/Scripts:/usr/local/bioinf/GeneMarkS-T:/home/gerid:/home/gerid/.local/bin:/home/gerid/perl5/bin:/usr/local/conda/envs/qiime2-2020.6/bin:/usr/local/conda/condabin:/usr/local/sbin:/usr/local/bin:/usr/sbin:/usr/bin:/sbin:/bin:/usr/local/conda/bin:/snap/bin

11

Maybe if I hide the old version of vsearch things will work smoothly?

12

Quick update. I commented out the line in my .profile containing the old vsearch, started a new session, and reran the code.
The code is still running (usually it would have exploded in red errors by now so thats good).
Stand by: The 'rocket' is still flying. I'll follow up with the first message from the line:

$ qiime vsearch join-pairs --i-demultiplexed-seqs demux_trimmed.qza --o-joined-sequences demux-joined.qza --p-minmergelen 300&

13

This PATH is a bit problematic - the general convention is an activated conda env should be first in the PATH, that way the packages in the env are loaded first when searching. I suspect you have manually added in your QIIME 2 2020.6 env into your path? You will possibly run into more odd errors in other places with QIIME 2, since the same overall problem exists here.

14

I see. I installed Qiime 2, 2020.6 the way its described on the Q2 website. I know no other way and only added things that I use on a regular to my path.

Correct me if im understanding this wrong, but if i rearrange the order in this path to have /usr/local/conda/envs/qiime2-2020.6/bin up front, i shouldn't have problems or need to comment out the other vsearch?

15

Yep - I don't think these two statements are mutually exclusive, right? The point is that your manual edits to your PATH are causing problems in your QIIME 2 env, and will likely be problematic elsewhere, too.

If you're using a standard miniconda or anaconda installation you shouldn't need to put this anywhere in your path manually - conda will add it for you automatically when you run conda activate qiime2-2020.6.

16

Thanks. I got vsearch to finish the run by commenting out the line in my path containing the old vsearch, however, I still dont quite understand how and where i manually screwed things up.
Does this mean that anytime I am messing with my path, i am potentially messing things up? I use vsearch for other applications aside from processing ASVs and OTUs via Q2, so having access to any vsearch in my path is useful.
Anyway, just some thoughts. Overall, Thank you so much for your help getting me over this hump and keeping me in the fight.

18

Hi @gollison!

No, not at all. The only thing that is "wrong" here is the order of the items in the PATH --- conda always expects to be first when an environment is active. If it isn't, then all kinds of bad things can happen.

Just set it lower in your path. If you don't have a conda env active, then the PATH will resolve on the custom installation location.

Hope that helps!

:qiime2:

20

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