I keep receiving an error from the DADA2 plugin suggesting that no reads are able to pass through the filter, but when looking at the quality plots, everything appears fairly normal. There are around 90 samples with an average of 128,489 reads per sample.
I have read the two other posts related to this error, it was recommended to try changing the length, this did not work for me. The forward and reverse reads are about 300 bp long and I tried truncating them at 250, 190 and 100 with no avail. It appears as the reads are fairly good quality so I'm not sure why this is happening. I've been able to pass this command before on a few different data sets, with similar looking quality plots.
It was also suggested to try using denoise-single instead of denoise-paired. This also didn't work for me and I again retrieved the same error. (The error I posted below is actually what was returned when I used Denoise-single).
I'm running this through qiime2-2018.11 on Cyverse Discovery Environment in Jupyter Notebook, and all commands leading up to the DADA2 Denoise step have worked.
Cyverse suggested using Deblur instead of DADA2, this also didn't work and another error was raised, but I would really like to use both my forward and reverse reads, so I would really prefer using dada2 denoise-paired if at all possible.
Commands ran
qiime tools import --type MultiplexedPairedEndBarcodeInSequence --input-path sequences --output-path paired-multiplexed.qza --input-format MultiplexedPairedEndBarcodeInSequenceDirFmt
qiime cutadapt demux-paired --i-seqs paired-multiplexed.qza --m-forward-barcodes-file barcodemap.txt --m-forward-barcodes-column BarcodeSequence --o-per-sample-sequences demultiplexed-seqs.qza --o-untrimmed-sequences untrimmed.qza
qiime cutadapt trim-paired --i-demultiplexed-sequences demultiplexed-seqs.qza --p-front-f GTAAAACGACGGCCAG --o-trimmed-sequences demux.qza --verbose
qiime demux summarize --i-data demux.qza --o-visualization demux.qzv
qiime dada2 denoise-paired --i-demultiplexed-seqs demux.qza --p-trim-left-f 13 --p-trim-left-r 13 --p-trunc-len-f 190 --p-trunc-len-r 190 --o-table table.qza --o-representative-sequences rep-seqs.qza --o-denoising-stats denoising-stats.qza --verbose
qiime dada2 denoise-single --i-demultiplexed-seqs demux.qza --p-trunc-len 190 --p-n-threads 0 --o-table table.qza --o-representative-sequences rep-seqs.qza --o-denoising-stats denoising-stats.qza --verbose
Error message
~/vice$ qiime dada2 denoise-single --i-demultiplexed-seqs demux.qza --p-trunc-len 190 --p-n-threads 0--o-table table.qza --o-representative-sequences rep-seqs.qza --o-denoising-stats denoising-stats.qza --verbose
Running external command line application(s). This may print messages to stdout and/or stderr.
The command(s) being run are below. These commands cannot be manually re-run as they will depend on temporary files that no longer exist.
Command: run_dada_single.R /var/tmp/q2-SingleLanePerSampleSingleEndFastqDirFmt-c9jltyfr /var/tmp/tmpp1yud6sq/output.tsv.biom /var/tmp/tmpp1yud6sq/track.tsv /var/tmp/tmpp1yud6sq 190 0 2.0 2 Inf consensus 1.0 0 1000000 NULL 16
Fatal error: cannot create 'R_TempDir'
Traceback (most recent call last):
File "/opt/conda/envs/qiime2-2018.11/lib/python3.5/site-packages/q2_dada2/_denoise.py", line 152, in _denoise_single
run_commands([cmd])
File "/opt/conda/envs/qiime2-2018.11/lib/python3.5/site-packages/q2_dada2/_denoise.py", line 36, in run_commands
subprocess.run(cmd, check=True)
File "/opt/conda/envs/qiime2-2018.11/lib/python3.5/subprocess.py", line 398, in run
output=stdout, stderr=stderr)
subprocess.CalledProcessError: Command '['run_dada_single.R', '/var/tmp/q2-SingleLanePerSampleSingleEndFastqDirFmt-c9jltyfr', '/var/tmp/tmpp1yud6sq/output.tsv.biom', '/var/tmp/tmpp1yud6sq/track.tsv', '/var/tmp/tmpp1yud6sq', '190', '0', '2.0', '2', 'Inf', 'consensus', '1.0', '0', '1000000', 'NULL', '16']' returned non-zero exit status 2
During handling of the above exception, another exception occurred:
Traceback (most recent call last):
File "/opt/conda/envs/qiime2-2018.11/lib/python3.5/site-packages/q2cli/commands.py", line 274, in call
results = action(**arguments)
File "", line 2, in denoise_single
File "/opt/conda/envs/qiime2-2018.11/lib/python3.5/site-packages/qiime2/sdk/action.py", line 231, in bound_callable
output_types, provenance)
File "/opt/conda/envs/qiime2-2018.11/lib/python3.5/site-packages/qiime2/sdk/action.py", line 362, in callable_executor
output_views = self._callable(**view_args)
File "/opt/conda/envs/qiime2-2018.11/lib/python3.5/site-packages/q2_dada2/_denoise.py", line 187, in denoise_single
band_size='16')
File "/opt/conda/envs/qiime2-2018.11/lib/python3.5/site-packages/q2_dada2/_denoise.py", line 159, in _denoise_single
" filter." % trunc_len)
ValueError: No reads passed the filter. trunc_len (190) may be longer than read lengths, or other arguments (such as max_ee or trunc_q) may be preventing reads from passing the filter.
Plugin error from dada2:
No reads passed the filter. trunc_len (190) may be longer than read lengths, or other arguments (such as max_ee or trunc_q) may be preventing reads from passing the filter.
See above for debug info.