hello,
I have encountered an error while running dada2 on a large number (1152) of 16S rRNA samples. It ran for about 3.5 days before it gave me an error, so I would like to trouble shoot what went wrong before I attempt to rerun it.
The sequencing data was obtained from JGI where each sample was returned as a single fastq file, which had already gone through some QC using BBDuk as follows:
"Sequence data for library was generated at the DOE Joint Genome Institute (JGI) using Illumina technology [1]. An Illumina library was constructed and sequenced 2x249 using the Illumina NovaSeq SP platform which generated 28,006 reads totaling 6,973,494 bp. BBDuk (version 38.90) [2] was used to remove contaminants [3], trim reads that contained adapter sequence and homopolymers of G's of size 5 or more at the ends of the reads and right quality trim reads where quality drops to 0. BBDuk was used to remove reads that contained 4 or more 'N' bases, had an average quality score across the read less than 3 or had a minimum length <= 51 bp or 33% of the full read length. Reads mapped with BBMap [2] to masked [5] human, cat, dog and mouse references at 93% identity were separated into a chaff file [4]. Reads aligned to common microbial contaminants [6] were separated into a chaff file [4]. The final filtered fastq contained 27,932 reads totaling 6,839,197 bp."
I imported the files into a QIIME2 artifact using:
qiime tools import
--type 'SampleData[SequencesWithQuality]'
--input-path Stands_Metadata.tsv
--output-path jgi-merged-demux.qza
--input-format SingleEndFastqManifestPhred33V2
After looking at the results of jgi-merged-demux.qza, I decided to run dada2 for further clean up steps as follows:
qiime dada2 denoise-single
--i-demultiplexed-seqs jgi-merged-demux.qza
--p-trim-left 7
--p-trunc-len 129
--o-representative-sequences rep-seqs-dada2.qza
--o-table table-dada2.qza
--o-denoising-stats stats-dada2.qza
After running for approximately 3.5 days, I encountered the following error:
"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/folders/jh/tpmtltq512vf0sx9n51fzxyw0000gn/T/qiime2-archive-3kytlkby/f34da75b-ed05-4076-80bf-60e09a2d9ce8/data /var/folders/jh/tpmtltq512vf0sx9n51fzxyw0000gn/T/tmptkpwp71b/output.tsv.biom /var/folders/jh/tpmtltq512vf0sx9n51fzxyw0000gn/T/tmptkpwp71b/track.tsv /var/folders/jh/tpmtltq512vf0sx9n51fzxyw0000gn/T/tmptkpwp71b 129 7 2.0 2 Inf independent consensus 1.0 1 1000000 NULL 16
R version 4.0.3 (2020-10-10)
Loading required package: Rcpp
DADA2: 1.18.0 / Rcpp: 1.0.6 / RcppParallel: 5.1.2
- Filtering ................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................
- Learning Error Rates
139425870 total bases in 1142835 reads from 2 samples will be used for learning the error rates. - Denoise samples .......................................................................................................................................................................................................................................................................................................................................................................Error in derepFastq(filts[[j]]) : Not all provided files exist.
Execution halted
Traceback (most recent call last):
File "/Users/mongomac/opt/miniconda3/envs/qiime2-2021.4/lib/python3.8/site-packages/q2_dada2/_denoise.py", line 182, in _denoise_single
run_commands([cmd])
File "/Users/mongomac/opt/miniconda3/envs/qiime2-2021.4/lib/python3.8/site-packages/q2_dada2/_denoise.py", line 36, in run_commands
subprocess.run(cmd, check=True)
File "/Users/mongomac/opt/miniconda3/envs/qiime2-2021.4/lib/python3.8/subprocess.py", line 516, in run
raise CalledProcessError(retcode, process.args,
subprocess.CalledProcessError: Command '['run_dada_single.R', '/var/folders/jh/tpmtltq512vf0sx9n51fzxyw0000gn/T/qiime2-archive-3kytlkby/f34da75b-ed05-4076-80bf-60e09a2d9ce8/data', '/var/folders/jh/tpmtltq512vf0sx9n51fzxyw0000gn/T/tmptkpwp71b/output.tsv.biom', '/var/folders/jh/tpmtltq512vf0sx9n51fzxyw0000gn/T/tmptkpwp71b/track.tsv', '/var/folders/jh/tpmtltq512vf0sx9n51fzxyw0000gn/T/tmptkpwp71b', '129', '7', '2.0', '2', 'Inf', 'independent', 'consensus', '1.0', '1', '1000000', 'NULL', '16']' returned non-zero exit status 1.
During handling of the above exception, another exception occurred:
Traceback (most recent call last):
File "/Users/mongomac/opt/miniconda3/envs/qiime2-2021.4/lib/python3.8/site-packages/q2cli/commands.py", line 329, in call
results = action(**arguments)
File "", line 2, in denoise_single
File "/Users/mongomac/opt/miniconda3/envs/qiime2-2021.4/lib/python3.8/site-packages/qiime2/sdk/action.py", line 244, in bound_callable
outputs = self.callable_executor(scope, callable_args,
File "/Users/mongomac/opt/miniconda3/envs/qiime2-2021.4/lib/python3.8/site-packages/qiime2/sdk/action.py", line 390, in callable_executor
output_views = self._callable(**view_args)
File "/Users/mongomac/opt/miniconda3/envs/qiime2-2021.4/lib/python3.8/site-packages/q2_dada2/_denoise.py", line 205, in denoise_single
return _denoise_single(
File "/Users/mongomac/opt/miniconda3/envs/qiime2-2021.4/lib/python3.8/site-packages/q2_dada2/_denoise.py", line 191, in _denoise_single
raise Exception("An error was encountered while running DADA2"
Exception: An error was encountered while running DADA2 in R (return code 1), please inspect stdout and stderr to learn more."
Please advise on what caused this error to occur and how to correct it.
I am running this on a Macbook Pro (mid 2014) running Mojave OS 10.14.6 with 2.2GHz Intel Core i7 processor and 16 GB 1600 MHz DDR3 memory.
QIIME2 version 2021.4 installed in a conda environment using command line interface (cli).
Thank you any advice or help you can provide!
Mike Ricketts