Hi everyone, hope you are well. I´m facing some issues while running the dada2 denoising step with my seq data. I have read similar topics in this forum but none with the same error.
My data come from Illumina MiSeq 300bp, PE data, is already demultiplexed and trimmed for adaptors and others using timmomatic:
for i1 in _R1; do i2=${i1/_R1/_R2}; java -jar /usr/share/java/trimmomatic-0.36.jar PE -phred33 -threads 8 $i1 i2 {i1/_R1/_R1_PE} {i1/_R1/_R1_UP} {i2/_R2/_R2_PE} ${i2/_R2/_R2_UP} ILLUMINACLIP:/usr/share/trimmomatic/TruSeq3-PE.fa:2:30:10 SLIDINGWINDOW:10:28 MINLEN:50 LEADING:8 TRAILING:8; done
After the trimmomatic run, I observed the resulting sequences and decided to run the in the dada2 denoisng step in order to trim the extremes tha still have low quality. The parameters I decided to use are the following:
(qiime2-2020.11) qiime2@qiime2core2020-11:/media/sf_qiime2/analisis_bac$ qiime dada2 denoise-paired --i-demultiplexed-seqs paired-end-demux.qza --p-trim-left-f 6 --p-trim-left-r 6 --p-trunc-len-f 289 --p-trunc-len-r 235 --o-table table16S.qza --o-representative-sequences rep_seqs16S.qza --o-denoising-stats denois_stats16S.qza --verbose
The error (or erros, I guess) is the following:
(qiime2-2020.11) qiime2@qiime2core2020-11:/media/sf_qiime2/analisis_bac$ qiime dada2 denoise-paired --i-demultiplexed-seqs paired-end-demux.qza --p-trim-left-f 6 --p-trim-left-r 6 --p-trunc-len-f 289 --p-trunc-len-r 235 --o-table table16S.qza --o-representative-sequences rep_seqs16S.qza --o-denoising-stats denois_stats16S.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/tmp7k45hvnp/forward /tmp/tmp7k45hvnp/reverse /tmp/tmp7k45hvnp/output.tsv.biom /tmp/tmp7k45hvnp/track.tsv /tmp/tmp7k45hvnp/filt_f /tmp/tmp7k45hvnp/filt_r 289 235 6 6 2.0 2.0 2 independent consensus 1.0 1 1000000
R version 4.0.2 (2020-06-22)
Loading required package: Rcpp
DADA2: 1.18.0 / Rcpp: 1.0.5 / RcppParallel: 5.0.2
- Filtering ........................
- Learning Error Rates
301954491 total bases in 1066977 reads from 11 samples will be used for learning the error rates.
Error in dada_uniques(names(drpi$uniques), unname(drpi$uniques), names(drpi$uniques) %in% :
Memory allocation failed.
Execution halted
Warning message:
system call failed: Cannot allocate memory
Traceback (most recent call last):
File "/home/qiime2/miniconda/envs/qiime2-2020.11/lib/python3.6/site-packages/q2_dada2/_denoise.py", line 264, in denoise_paired
run_commands([cmd])
File "/home/qiime2/miniconda/envs/qiime2-2020.11/lib/python3.6/site-packages/q2_dada2/_denoise.py", line 36, in run_commands
subprocess.run(cmd, check=True)
File "/home/qiime2/miniconda/envs/qiime2-2020.11/lib/python3.6/subprocess.py", line 438, in run
output=stdout, stderr=stderr)
subprocess.CalledProcessError: Command '['run_dada_paired.R', '/tmp/tmp7k45hvnp/forward', '/tmp/tmp7k45hvnp/reverse', '/tmp/tmp7k45hvnp/output.tsv.biom', '/tmp/tmp7k45hvnp/track.tsv', '/tmp/tmp7k45hvnp/filt_f', '/tmp/tmp7k45hvnp/filt_r', '289', '235', '6', '6', '2.0', '2.0', '2', 'independent', '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/qiime2/miniconda/envs/qiime2-2020.11/lib/python3.6/site-packages/q2cli/commands.py", line 329, in call
results = action(**arguments)
File "", line 2, in denoise_paired
File "/home/qiime2/miniconda/envs/qiime2-2020.11/lib/python3.6/site-packages/qiime2/sdk/action.py", line 245, in bound_callable
output_types, provenance)
File "/home/qiime2/miniconda/envs/qiime2-2020.11/lib/python3.6/site-packages/qiime2/sdk/action.py", line 390, in callable_executor
output_views = self._callable(**view_args)
File "/home/qiime2/miniconda/envs/qiime2-2020.11/lib/python3.6/site-packages/q2_dada2/_denoise.py", line 279, 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.
(qiime2-2020.11) qiime2@qiime2core2020-11:/media/sf_qiime2/analisis_bac$
Any suggestion for what is happening here will be very useful!!!
Thank you!