So as a test I used just a single sample and I concatenated the fw and rv reads. That ran through the import fine. I did get an usual comment on the interactive quality plot (see file attached). The comment was in red and said "The plot at position 157 was generated using a random sampling of 5071 out of 1475320 sequences without replacement. This position (157) is greater than the minimum sequence length observed during subsampling (58 bases). As a result, the plot at this position is not based on data from all of the sequences, so it should be interpreted with caution when compared to plots for other positions. Outlier quality scores are not shown in box plots for clarity." Any help on the dummy version of what this is saying?
Secondly, when I tried to use dada2 denoise on this sequence just as a test I got an error that I don't quite understand either. It is the following and its long so I apologize for that:
MY COMMAND: qiime dada2 denoise-paired --i-demultiplexed-seqs demux-paired-end.qza --p-trim-left-f 0 --p-trunc-len-f 200 --p-trim-left-r 0 --p-trunc-len-r 200 --o-representative-sequences rep-seqs-dada2.250.qza --o-table table-dada2.250.qza --p-n-threads 16 --o-denoising-stats denoising-stats-dada2-250.qza --verbose
ERROR AS GIVEN BY --VERBOSE COMMAND:
R version 3.4.1 (2017-06-30)
Loading required package: Rcpp
DADA2 R package version: 1.6.0
- Filtering Error in add(bin) : internal: buf !=
Traceback (most recent call last):
File "/usr/local/packages/qiime2/2018.4/lib/python3.5/site-packages/q2_dada2/_denoise.py", line 229, in denoise_paired
File "/usr/local/packages/qiime2/2018.4/lib/python3.5/site-packages/q2_dada2/_denoise.py", line 36, in run_commands
File "/usr/local/packages/qiime2/2018.4/lib/python3.5/subprocess.py", line 398, in run
subprocess.CalledProcessError: Command '['run_dada_paired.R', '/tmp/tmpsad_b3h3/forward', '/tmp/tmpsad_b3h3/reverse', '/tmp/tmpsad_b3h3/output.tsv.biom', '/tmp/tmpsad_b3h3/track.tsv', '/tmp/tmpsad_b3h3/filt_f', '/tmp/tmpsad_b3h3/filt_r', '200', '200', '0', '0', '2.0', '2', 'consensus', '1.0', '16', '1000000']' returned non-zero exit status 1
During handling of the above exception, another exception occurred:
Traceback (most recent call last):
File "/usr/local/packages/qiime2/2018.4/lib/python3.5/site-packages/q2cli/commands.py", line 274, in call
results = action(**arguments)
File "", line 2, in denoise_paired
File "/usr/local/packages/qiime2/2018.4/lib/python3.5/site-packages/qiime2/sdk/action.py", line 231, in bound_callable
File "/usr/local/packages/qiime2/2018.4/lib/python3.5/site-packages/qiime2/sdk/action.py", line 366, in callable_executor
output_views = self._callable(**view_args)
File "/usr/local/packages/qiime2/2018.4/lib/python3.5/site-packages/q2_dada2/_denoise.py", line 244, in denoise_paired
" and stderr to learn more." % e.returncode)
Exception: An error was encountered while running DADA2 in R (return code 1), please inspect stdout and stderr to learn more.
Plugin error from dada2:
An error was encountered while running DADA2 in R (return code 1), please inspect stdout and stderr to learn more.
See above for debug info.
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_paired.R /tmp/tmpsad_b3h3/forward /tmp/tmpsad_b3h3/reverse /tmp/tmpsad_b3h3/output.tsv.biom /tmp/tmpsad_b3h3/track.tsv /tmp/tmpsad_b3h3/filt_f /tmp/tmpsad_b3h3/filt_r 200 200 0 0 2.0 2 consensus 1.0 16 1000000
Are these two errors somehow related? If they are any idea what might be causing them? This is just from a single sample, single fw and single rv fastq. I did concatenate them by hand, but that shouldn't be a huge issue I don't think. I was pretty careful when I did it.
Any advice on how to get this fixed?
demux.qzv (266.9 KB)