Hello, I apologise if this has been answered but I searched for this error and saw it has been answered previously, though my error seems to be different from others. I am getting an error running dada2 denoising with my demultiplexed data (16S metabarcoding data). This data was demultiplexed by the sequencing centre, and then I imported the data, trimmed primers, and tried dada2 denoising.
I am using QIIME2-2022.2 and will post my import through dada2 commands, followed by a verbose output from running my denoising commands. My sequence files are gzipped fastq's with names like 16S_485686R1.fastq.gz and the same R2 names. Any help deciphering the error would be very much appreciated.
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conda activate qiime2-2022.2
#check currently active conda environment
conda info
#activate tab completion
source tab-qiime
#Now import our data using a 'manifest' file of all fastq file names
qiime tools import
--type 'SampleData[PairedEndSequencesWithQuality]'
--input-path pe33-16Smanifest
--input-format PairedEndFastqManifestPhred33V2
--output-path 16S-combined-demux.qza
#Now trim primers
qiime cutadapt trim-paired
--i-demultiplexed-sequences 16S-combined-demux.qza
--p-front-f AGCGYAATCACTTGTCTYTTAA
--p-front-r CRBGGTCGCCCCAACCRAA
--p-error-rate 0.11
--output-dir trimmed
--verbose
qiime dada2 denoise-paired \
--i-demultiplexed-seqs trimmed/trimmed_sequences.qza
--p-trunc-len-f 158
--p-trunc-len-r 158
--p-n-threads 0
--p-min-overlap 12
--p-pooling-method independent
--output-dir trimmed/dada2out
--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/tmpv3r6rmfb/forward /tmp/tmpv3r6rmfb/reverse /tmp/tmpv3r6rmfb/output.tsv.biom /tmp/tmpv3r6rmfb/track.tsv /tmp/tmpv3r6rmfb/filt_f /tmp/tmpv3r6rmfb/filt_r 158 158 0 0 2.0 2.0 2 12 independent consensus 1.0 0 1000000
R version 4.1.3 (2022-03-10)
Loading required package: Rcpp
DADA2: 1.22.0 / Rcpp: 1.0.8.3 / RcppParallel: 5.1.5
- Filtering The filter removed all reads: /tmp/tmpv3r6rmfb/filt_f/Sample-111_110_L001_R1_001.fastq.gz and /tmp/tmpv3r6rmfb/filt_r/Sample-111_225_L001_R2_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpv3r6rmfb/filt_f/Sample-115_114_L001_R1_001.fastq.gz and /tmp/tmpv3r6rmfb/filt_r/Sample-115_229_L001_R2_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpv3r6rmfb/filt_f/Sample-114_113_L001_R1_001.fastq.gz and /tmp/tmpv3r6rmfb/filt_r/Sample-114_228_L001_R2_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpv3r6rmfb/filt_f/Sample-104_103_L001_R1_001.fastq.gz and /tmp/tmpv3r6rmfb/filt_r/Sample-104_218_L001_R2_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpv3r6rmfb/filt_f/Sample-3_2_L001_R1_001.fastq.gz and /tmp/tmpv3r6rmfb/filt_r/Sample-3_117_L001_R2_001.fastq.gz not written.
The filter removed all reads: /tmp/tmpv3r6rmfb/filt_f/Sample-96_95_L001_R1_001.fastq.gz and /tmp/tmpv3r6rmfb/filt_r/Sample-96_210_L001_R2_001.fastq.gz not written.
Some input samples had no reads pass the filter.
......x.......x..xx...................x........................................................................x... - Learning Error Rates
231020332 total bases in 1462154 reads from 2 samples will be used for learning the error rates.
231020332 total bases in 1462154 reads from 2 samples will be used for learning the error rates.
Error rates could not be estimated (this is usually because of very few reads).
Error in getErrors(err, enforce = TRUE) : Error matrix is NULL.
Execution halted
Traceback (most recent call last):
File "/home/mcrg/Downloads/usr/bin/envs/qiime2-2022.2/lib/python3.8/site-packages/q2_dada2/_denoise.py", line 279, in denoise_paired
run_commands([cmd])
File "/home/mcrg/Downloads/usr/bin/envs/qiime2-2022.2/lib/python3.8/site-packages/q2_dada2/_denoise.py", line 36, in run_commands
subprocess.run(cmd, check=True)
File "/home/mcrg/Downloads/usr/bin/envs/qiime2-2022.2/lib/python3.8/subprocess.py", line 516, in run
raise CalledProcessError(retcode, process.args,
subprocess.CalledProcessError: Command '['run_dada_paired.R', '/tmp/tmpv3r6rmfb/forward', '/tmp/tmpv3r6rmfb/reverse', '/tmp/tmpv3r6rmfb/output.tsv.biom', '/tmp/tmpv3r6rmfb/track.tsv', '/tmp/tmpv3r6rmfb/filt_f', '/tmp/tmpv3r6rmfb/filt_r', '158', '158', '0', '0', '2.0', '2.0', '2', '12', 'independent', 'consensus', '1.0', '0', '1000000']' returned non-zero exit status 1.
During handling of the above exception, another exception occurred:
Traceback (most recent call last):
File "/home/mcrg/Downloads/usr/bin/envs/qiime2-2022.2/lib/python3.8/site-packages/q2cli/commands.py", line 339, in call
results = action(**arguments)
File "", line 2, in denoise_paired
File "/home/mcrg/Downloads/usr/bin/envs/qiime2-2022.2/lib/python3.8/site-packages/qiime2/sdk/action.py", line 245, in bound_callable
outputs = self.callable_executor(scope, callable_args,
File "/home/mcrg/Downloads/usr/bin/envs/qiime2-2022.2/lib/python3.8/site-packages/qiime2/sdk/action.py", line 391, in callable_executor
output_views = self._callable(**view_args)
File "/home/mcrg/Downloads/usr/bin/envs/qiime2-2022.2/lib/python3.8/site-packages/q2_dada2/_denoise.py", line 292, in denoise_paired
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.
Plugin error from dada2:
An error was encountered while running DADA2 in R (return code 1), please inspect stdout and stderr to learn more.