Hello everyone,
I am working with paired-end sequences of 16S V3-V4 region (341F/805R primers) from lake samples (Illumina MiSeq). I have removed primers from the sequences using cutadapt software
antartida-paired-end.cutadapt.qzv (291.9 KB)
After that, I have run DADA2 in order to denoise sequences and I obtained the following error (commands):
qiime dada2 denoise-paired
--i-demultiplexed-seqs antartida-paired-end.cutadapt.qza
--p-trim-left-f 0
--p-trunc-len-f 290
--p-trim-left-r 0
--p-trunc-len-r 290
--o-representative-sequences rep-seq-antartida.cutadapted.qza
--o-table table-antartida.cutadapted.qza
--o-denoising-stats stats-dada2.antartida.cutadapted.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_paired.R /tmp/tmpggjcg2sa/forward /tmp/tmpggjcg2sa/reverse /tmp/tmpggjcg2sa/output.tsv.biom /tmp/tmpggjcg2sa/track.tsv /tmp/tmpggjcg2sa/filt_f /tmp/tmpggjcg2sa/filt_r 290 290 0 0 2.0 2 consensus 1.0 1 1000000
R version 3.4.1 (2017-06-30)
Loading required package: Rcpp
DADA2 R package version: 1.6.0
-
Filtering The filter removed all reads: /tmp/tmpggjcg2sa/filt_f/91_0_L001_R1_001.fastq.gz and /tmp/tmpggjcg2sa/filt_r/91_1_L001_R2_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpggjcg2sa/filt_f/A2_2_L001_R1_001.fastq.gz and /tmp/tmpggjcg2sa/filt_r/A2_3_L001_R2_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpggjcg2sa/filt_f/C1_4_L001_R1_001.fastq.gz and /tmp/tmpggjcg2sa/filt_r/C1_5_L001_R2_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpggjcg2sa/filt_f/DNA_10_L001_R1_001.fastq.gz and /tmp/tmpggjcg2sa/filt_r/DNA_11_L001_R2_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpggjcg2sa/filt_f/Q_12_L001_R1_001.fastq.gz and /tmp/tmpggjcg2sa/filt_r/Q_13_L001_R2_001.fastq.gz not written.
Some input samples had no reads pass the filter.
xxx..xx -
Learning Error Rates
2a) Forward Reads
Initializing error rates to maximum possible estimate.
Sample 1 - 1 reads in 1 unique sequences.
Sample 2 - 1 reads in 1 unique sequences.
selfConsist step 2
Convergence after 2 rounds.
2b) Reverse Reads
Initializing error rates to maximum possible estimate.
Sample 1 - 1 reads in 1 unique sequences.
Sample 2 - 1 reads in 1 unique sequences.
selfConsist step 2
Convergence after 2 rounds. -
Denoise remaining samples
-
Remove chimeras (method = consensus)
Error in isBimeraDenovoTable(unqs[[i]], ..., verbose = verbose) :
Input must be a valid sequence table.
Calls: removeBimeraDenovo -> isBimeraDenovoTable
Además: Warning message:
In is.na(colnames(unqs[[i]])) :
is.na() aplicado a un objeto que no es (lista o vector) de tipo 'NULL
EjecuciĂłn interrumpida
Traceback (most recent call last):
File "/home/guille/miniconda3/envs/qiime2-2019.1/lib/python3.6/site-packages/q2_dada2/_denoise.py", line 231, in denoise_paired
run_commands([cmd])
File "/home/guille/miniconda3/envs/qiime2-2019.1/lib/python3.6/site-packages/q2_dada2/_denoise.py", line 36, in run_commands
subprocess.run(cmd, check=True)
File "/home/guille/miniconda3/envs/qiime2-2019.1/lib/python3.6/subprocess.py", line 418, in run
output=stdout, stderr=stderr)
subprocess.CalledProcessError: Command '['run_dada_paired.R', '/tmp/tmpggjcg2sa/forward', '/tmp/tmpggjcg2sa/reverse', '/tmp/tmpggjcg2sa/output.tsv.biom', '/tmp/tmpggjcg2sa/track.tsv', '/tmp/tmpggjcg2sa/filt_f', '/tmp/tmpggjcg2sa/filt_r', '290', '290', '0', '0', '2.0', '2', 'consensus', '1.0', '1', '1000000']' returned non-zero exit status 1.
During handling of the above exception, another exception occurred:
Traceback (most recent call last):
File "/home/guille/miniconda3/envs/qiime2-2019.1/lib/python3.6/site-packages/q2cli/commands.py", line 274, in call
results = action(**arguments)
File "</home/guille/miniconda3/envs/qiime2-2019.1/lib/python3.6/site-packages/decorator.py:decorator-gen-442>", line 2, in denoise_paired
File "/home/guille/miniconda3/envs/qiime2-2019.1/lib/python3.6/site-packages/qiime2/sdk/action.py", line 231, in bound_callable
output_types, provenance)
File "/home/guille/miniconda3/envs/qiime2-2019.1/lib/python3.6/site-packages/qiime2/sdk/action.py", line 365, in callable_executor
output_views = self._callable(**view_args)
File "/home/guille/miniconda3/envs/qiime2-2019.1/lib/python3.6/site-packages/q2_dada2/_denoise.py", line 246, 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.
It seems that there is a problem with my sequences because the filter removed alls reads in all samples. I have tested DADA2 denoising step in sequences prior primer trimming with cutadapt and it worked. I dont know where is the error. Anybody could help me? I use QIIME2 2019.1 in a linux mint distro. Thank you in advance.
Guille
