Hi! I am still relatively new to qiime2 but am running into this error I've not seen before as well as cannot figure out why this error would be occurring. I am running Qiime2 Core 2019.4 in a Virtualbox. My sequences are 16S rRNA sequenced generated by an illuminia Miseq and I am running my demultiplexed single-end 16S sequences through the dada2 denoise-single command and receiving the following plugin error from dada2, my code (run with verbose) and error copied below.
(qiime2-2019.4) qiime2@qiime2core2019-4:~/AMD2_9.19$ qiime dada2 denoise-single --i-demultiplexed-seqs demux.qza --p-trim-left 0 --p-trunc-len 220 --o-representative-sequences re-seqs-dada2.qza --o-table table-dada2.qza --o-denoising-stats stats-dada2.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 /tmp/qiime2-archive-ywmanxjw/ad572a07-92ae-4a6f-b0df-76e262b4793a/data /tmp/tmpog_3cban/output.tsv.biom /tmp/tmpog_3cban/track.tsv /tmp/tmpog_3cban 220 0 2.0 2 Inf consensus 1.0 1 1000000 NULL 16
R version 3.5.1 (2018-07-02)
Loading required package: Rcpp
DADA2: 1.10.0 / Rcpp: 1.0.1 / RcppParallel: 4.4.2
- Filtering The filter removed all reads: /tmp/tmpog_3cban/1wk_8_L001_R1_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpog_3cban/2wk_5_L001_R1_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpog_3cban/4wk_2_L001_R1_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpog_3cban/6wk_4_L001_R1_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpog_3cban/Dup_7_L001_R1_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpog_3cban/NoCore_1_L001_R1_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpog_3cban/Raw1_3_L001_R1_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpog_3cban/Raw2_6_L001_R1_001.fastq.gz not written.
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Error: No reads passed the filter (was truncLen longer than the read length?)
Traceback (most recent call last):
File "/home/qiime2/miniconda/envs/qiime2-2019.4/lib/python3.6/site-packages/q2_dada2/_denoise.py", line 152, in _denoise_single
run_commands([cmd])
File "/home/qiime2/miniconda/envs/qiime2-2019.4/lib/python3.6/site-packages/q2_dada2/_denoise.py", line 36, in run_commands
subprocess.run(cmd, check=True)
File "/home/qiime2/miniconda/envs/qiime2-2019.4/lib/python3.6/subprocess.py", line 418, in run
output=stdout, stderr=stderr)
subprocess.CalledProcessError: Command '['run_dada_single.R', '/tmp/qiime2-archive-ywmanxjw/ad572a07-92ae-4a6f-b0df-76e262b4793a/data', '/tmp/tmpog_3cban/output.tsv.biom', '/tmp/tmpog_3cban/track.tsv', '/tmp/tmpog_3cban', '220', '0', '2.0', '2', 'Inf', 'consensus', '1.0', '1', '1000000', 'NULL', '16']' returned non-zero exit status 2.
During handling of the above exception, another exception occurred:
Traceback (most recent call last):
File "/home/qiime2/miniconda/envs/qiime2-2019.4/lib/python3.6/site-packages/q2cli/commands.py", line 311, in call
results = action(**arguments)
File "</home/qiime2/miniconda/envs/qiime2-2019.4/lib/python3.6/site-packages/decorator.py:decorator-gen-449>", line 2, in denoise_single
File "/home/qiime2/miniconda/envs/qiime2-2019.4/lib/python3.6/site-packages/qiime2/sdk/action.py", line 231, in bound_callable
output_types, provenance)
File "/home/qiime2/miniconda/envs/qiime2-2019.4/lib/python3.6/site-packages/qiime2/sdk/action.py", line 365, in callable_executor
output_views = self._callable(**view_args)
File "/home/qiime2/miniconda/envs/qiime2-2019.4/lib/python3.6/site-packages/q2_dada2/_denoise.py", line 187, in denoise_single
band_size='16')
File "/home/qiime2/miniconda/envs/qiime2-2019.4/lib/python3.6/site-packages/q2_dada2/_denoise.py", line 159, in _denoise_single
" filter." % trunc_len)
ValueError: No reads passed the filter. trunc_len (220) 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 (220) 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.
Debug info has been saved to /tmp/qiime2-q2cli-err-5vdq9n9r.log. No reads passed the filter. trunc_len (220) may be longer than read lengths, or other arguments (such as max_ee or trunc_q) may be preventing reads from passing the filter.
Debug info is uploaded here as a .txt qiime2-q2cli-err-5vdq9n9r.txt (3.3 KB)
My demux.qza file is here. I have 8 samples and my sequences are successfully demultiplexed and are all at a length of 251 bases, as can be seen in the below quality plot and viewed using qiime tools view demux.qzv therefore there should not be an issue with the truncated length being longer than my actual reads. I've tried trimming to several length including 0, 50, 120, 220 and receive the same error every time.
I have checked other posts from qiime users who have received this error however they are using forward and reverse reads and their issue seems to deal more with joining reads that are too short, in my case the reads should be long enough as the demux file contains the demultiplexed reads with 251 bp.
Thank you so much, I've tried several different things and can't seem to diagnose the problem, I appreciate any insight and guidance.