Dear all,
I am trying to combine sequence data from two runs A and B. Data in A and B are unmerged fastq read pairs R1 and R2. I am describing the problem breifly here, it’s an odd one. Despite my short post I would be extremely grateful to hear an opinion on this. Running Qiime 2017.12.
-
Processing for run
A(completes successfully)- Spliting
.fastqfiles by primers (to retain only 18S reads in mixed-loci libraries with identical barcodes: Tried this output during analysisA&B- fails - trimming using
trimmomatic: Tried this output during analysisA&B- fails qiime tools importqiime2cli demux summarizeqiime2cli dada2 denoise-paired- …
- Spliting
-
Processing for run
B(completes successfully)qiime2cli tools importqiime cutadapt trim-pairedqiime2cli demux summarizeqiime2cli metadata tabulateqiime2cli dada2 denoise-paired
-
Processing for run
A&Bqiime2cli tools import-
qiime cutadapt trim-paired… fails - see error messages below - parts ofR1andR2are “improperly paired”. Any one any ideas how this can happen, has this happened to anyone before.
I would be looking forward to hearing of your experiences and opinions.
Kind regards and thanks for working on Qiime,
Paul
Error messages
(a.k.a computer vomit)
++ qiime cutadapt trim-paired --i-demultiplexed-sequences /Users/paul/Documents/CU_combined/Zenodo/Qiime/040_18S_paired-end-import.qza --p-cores 2 --p-front-f GCGGTAATTCCAGCTCCAA --p-f
ront-r TTGGCAAATGCTTTCGC --p-error-rate 0.2 --o-trimmed-sequences /Users/paul/Documents/CU_combined/Zenodo/Qiime/042_18S_paired-end-trimmed.qza --verbose
Running external command line application. This may print messages to stdout and/or stderr.
The commands to be run are below. These commands cannot be manually re-run as they will depend on temporary files that no longer exist.
Command: cutadapt --cores 2 --error-rate 0.2 --times 1 --overlap 3 -o /var/folders/jh/3gz6s81d2x7gjd30dcywtqpw0000gn/T/q2-CasavaOneEightSingleLanePerSampleDirFmt-oi0k6eyg/CH00-0301_62_
L001_R1_001.fastq.gz -p /var/folders/jh/3gz6s81d2x7gjd30dcywtqpw0000gn/T/q2-CasavaOneEightSingleLanePerSampleDirFmt-oi0k6eyg/CH00-0301_63_L001_R2_001.fastq.gz --front GCGGTAATTCCAGCTCC
AA -G TTGGCAAATGCTTTCGC /var/folders/jh/3gz6s81d2x7gjd30dcywtqpw0000gn/T/qiime2-archive-zxavzj1i/59b9a374-550a-45f7-9467-5b22262183e3/data/CH00-0301_62_L001_R1_001.fastq.gz /var/folder
s/jh/3gz6s81d2x7gjd30dcywtqpw0000gn/T/qiime2-archive-zxavzj1i/59b9a374-550a-45f7-9467-5b22262183e3/data/CH00-0301_63_L001_R2_001.fastq.gz
This is cutadapt 1.15 with Python 3.5.4
Command line parameters: --cores 2 --error-rate 0.2 --times 1 --overlap 3 -o /var/folders/jh/3gz6s81d2x7gjd30dcywtqpw0000gn/T/q2-CasavaOneEightSingleLanePerSampleDirFmt-oi0k6eyg/CH00-0
301_62_L001_R1_001.fastq.gz -p /var/folders/jh/3gz6s81d2x7gjd30dcywtqpw0000gn/T/q2-CasavaOneEightSingleLanePerSampleDirFmt-oi0k6eyg/CH00-0301_63_L001_R2_001.fastq.gz --front GCGGTAATTC
CAGCTCCAA -G TTGGCAAATGCTTTCGC /var/folders/jh/3gz6s81d2x7gjd30dcywtqpw0000gn/T/qiime2-archive-zxavzj1i/59b9a374-550a-45f7-9467-5b22262183e3/data/CH00-0301_62_L001_R1_001.fastq.gz /var
/folders/jh/3gz6s81d2x7gjd30dcywtqpw0000gn/T/qiime2-archive-zxavzj1i/59b9a374-550a-45f7-9467-5b22262183e3/data/CH00-0301_63_L001_R2_001.fastq.gz
Running on 2 cores
Trimming 2 adapters with at most 20.0% errors in paired-end mode ...
ERROR: Traceback (most recent call last):
File "/Applications/miniconda3/envs/qiime2-2017.12/lib/python3.5/site-packages/cutadapt/pipeline.py", line 454, in run
(n, bp1, bp2) = self._pipeline.process_reads()
File "/Applications/miniconda3/envs/qiime2-2017.12/lib/python3.5/site-packages/cutadapt/pipeline.py", line 282, in process_reads
ork/Frameworks/Metadata.framework/Versions/A/Supp
for read1, read2 in self._reader:
File "/Applications/miniconda3/envs/qiime2-2017.12/lib/python3.5/site-packages/cutadapt/seqio.py", line 420, in __iter__
"in file 1 does not match '{1}' in file 2.".format(r1.name, r2.name))
cutadapt.seqio.FormatError: Reads are improperly paired. Read name 'M00314:113:000000000-AJW1N:1:1111:23432:12094 1:N:0:1' in file 1 does not match 'M00314:113:000000000-AJW1N:1:1101:1
8027:1086 2:N:0:1' in file 2.
cutadapt: error: Reads are improperly paired. Read name 'M00314:113:000000000-AJW1N:1:1111:23432:12094 1:N:0:1' in file 1 does not match 'M00314:113:000000000-AJW1N:1:1101:18027:1086 2
:N:0:1' in file 2.
Traceback (most recent call last):
onda3/envs/qiime2-2017.12/lib/python3.5/site-packages/q2cli/commands.py", line 224, in __call__ │
results = action(**arguments)
File "<decorator-gen-358>", line 2, in trim_paired
File "/Applications/miniconda3/envs/qiime2-2017.12/lib/python3.5/site-packages/qiime2/sdk/action.py", line 228, in bound_callable
output_types, provenance)
File "/Applications/miniconda3/envs/qiime2-2017.12/lib/python3.5/site-packages/qiime2/sdk/action.py", line 363, in _callable_executor_
output_views = self._callable(**view_args)
File "/Applications/miniconda3/envs/qiime2-2017.12/lib/python3.5/site-packages/q2_cutadapt/_trim.py", line 172, in trim_paired
run_commands(cmds)
File "/Applications/miniconda3/envs/qiime2-2017.12/lib/python3.5/site-packages/q2_cutadapt/_trim.py", line 28, in run_commands
subprocess.run(cmd, check=True)
File "/Applications/miniconda3/envs/qiime2-2017.12/lib/python3.5/subprocess.py", line 398, in run
output=stdout, stderr=stderr)
subprocess.CalledProcessError: Command '['cutadapt', '--cores', '2', '--error-rate', '0.2', '--times', '1', '--overlap', '3', '-o', '/var/folders/jh/3gz6s81d2x7gjd30dcywtqpw0000gn/T/q2
-CasavaOneEightSingleLanePerSampleDirFmt-oi0k6eyg/CH00-0301_62_L001_R1_001.fastq.gz', '-p', '/var/folders/jh/3gz6s81d2x7gjd30dcywtqpw0000gn/T/q2-CasavaOneEightSingleLanePerSampleDirFmt
-oi0k6eyg/CH00-0301_63_L001_R2_001.fastq.gz', '--front', 'GCGGTAATTCCAGCTCCAA', '-G', 'TTGGCAAATGCTTTCGC', '/var/folders/jh/3gz6s81d2x7gjd30dcywtqpw0000gn/T/qiime2-archive-zxavzj1i/59b
9a374-550a-45f7-9467-5b22262183e3/data/CH00-0301_62_L001_R1_001.fastq.gz', '/var/folders/jh/3gz6s81d2x7gjd30dcywtqpw0000gn/T/qiime2-archive-zxavzj1i/59b9a374-550a-45f7-9467-5b22262183e
3/data/CH00-0301_63_L001_R2_001.fastq.gz']' returned non-zero exit status 1
Plugin error from cutadapt:
Command '['cutadapt', '--cores', '2', '--error-rate', '0.2', '--times', '1', '--overlap', '3', '-o', '/var/folders/jh/3gz6s81d2x7gjd30dcywtqpw0000gn/T/q2-CasavaOneEightSingleLanePerS
ampleDirFmt-oi0k6eyg/CH00-0301_62_L001_R1_001.fastq.gz', '-p', '/var/folders/jh/3gz6s81d2x7gjd30dcywtqpw0000gn/T/q2-CasavaOneEightSingleLanePerSampleDirFmt-oi0k6eyg/CH00-0301_63_L001_R
2_001.fastq.gz', '--front', 'GCGGTAATTCCAGCTCCAA', '-G', 'TTGGCAAATGCTTTCGC', '/var/folders/jh/3gz6s81d2x7gjd30dcywtqpw0000gn/T/qiime2-archive-zxavzj1i/59b9a374-550a-45f7-9467-5b222621
83e3/data/CH00-0301_62_L001_R1_001.fastq.gz', '/var/folders/jh/3gz6s81d2x7gjd30dcywtqpw0000gn/T/qiime2-archive-zxavzj1i/59b9a374-550a-45f7-9467-5b22262183e3/data/CH00-0301_63_L001_R2_0
01.fastq.gz']' returned non-zero exit status 1
See above for debug info.
