Hi there,
Im trying to perform an Alpha diversity analysis using the "Moving Pictures" guide and it says to use DADA2 to form a feature table. I do so but I receive an error message as seen below.
(qiime2-2022.11) [email protected]:~/Documents/Test/raw_read_files/7HB$ qiime dada2 denoise-single --i-demultiplexed-seqs 7HBDemux.qza --p-trunc-len 0 --o-table 7HBTable.qza --o-representative-sequences 7HBRepresentativeSequences.qza --o-denoising-stats 7HBDenoiseStats.qza
Plugin error from dada2:An error was encountered while running DADA2 in R (return code 1), please inspect stdout and stderr to learn more.
Debug info has been saved to /tmp/qiime2-q2cli-err-n27cy5g5.log
And the error message is:
(qiime2-2022.11) [email protected]:~/Documents/Test/raw_read_files/7HB$ cat /tmp/qiime2-q2cli-err-w618n7xs.log
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.R --input_directory /tmp/qiime2/fa00899/data/eb869357-315f-4370-85c0-4d3d9f2e5123/data --output_path /tmp/tmpbr4gobd6/output.tsv.biom --output_track /tmp/tmpbr4gobd6/track.tsv --filtered_directory /tmp/tmpbr4gobd6 --truncation_length 0 --trim_left 0 --max_expected_errors 2.0 --truncation_quality_score 2 --max_length Inf --pooling_method independent --chimera_method consensus --min_parental_fold 1.0 --allow_one_off False --num_threads 1 --learn_min_reads 1000000 --homopolymer_gap_penalty NULL --band_size 16
R version 4.2.2 (2022-10-31)
Loading required package: Rcpp
DADA2: 1.26.0 / Rcpp: 1.0.9 / RcppParallel: 5.1.6
2) Filtering .
3) Learning Error Rates
17782172 total bases in 54338 reads from 1 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.
6: stop("Error matrix is NULL.")
5: getErrors(err, enforce = TRUE)
4: dada(drps, err = NULL, errorEstimationFunction = errorEstimationFunction,
selfConsist = TRUE, multithread = multithread, verbose = verbose,
MAX_CONSIST = MAX_CONSIST, OMEGA_C = OMEGA_C, ...)
3: learnErrors(filts, nreads = nreads.learn, multithread = multithread,
HOMOPOLYMER_GAP_PENALTY = HOMOPOLYMER_GAP_PENALTY, BAND_SIZE = BAND_SIZE)
2: withCallingHandlers(expr, warning = function(w) if (inherits(w,
classes)) tryInvokeRestart("muffleWarning"))
1: suppressWarnings(learnErrors(filts, nreads = nreads.learn, multithread = multithread,
HOMOPOLYMER_GAP_PENALTY = HOMOPOLYMER_GAP_PENALTY, BAND_SIZE = BAND_SIZE))
Traceback (most recent call last):
File "/user/HS501/fa00899/miniconda3/envs/qiime2-2022.11/lib/python3.8/site-packages/q2_dada2/_denoise.py", line 220, in _denoise_single
run_commands([cmd])
File "/user/HS501/fa00899/miniconda3/envs/qiime2-2022.11/lib/python3.8/site-packages/q2_dada2/_denoise.py", line 36, in run_commands
subprocess.run(cmd, check=True)
File "/user/HS501/fa00899/miniconda3/envs/qiime2-2022.11/lib/python3.8/subprocess.py", line 516, in run
raise CalledProcessError(retcode, process.args,
subprocess.CalledProcessError: Command '['run_dada.R', '--input_directory', '/tmp/qiime2/fa00899/data/eb869357-315f-4370-85c0-4d3d9f2e5123/data', '--output_path', '/tmp/tmpbr4gobd6/output.tsv.biom', '--output_track', '/tmp/tmpbr4gobd6/track.tsv', '--filtered_directory', '/tmp/tmpbr4gobd6', '--truncation_length', '0', '--trim_left', '0', '--max_expected_errors', '2.0', '--truncation_quality_score', '2', '--max_length', 'Inf', '--pooling_method', 'independent', '--chimera_method', 'consensus', '--min_parental_fold', '1.0', '--allow_one_off', 'False', '--num_threads', '1', '--learn_min_reads', '1000000', '--homopolymer_gap_penalty', 'NULL', '--band_size', '16']' returned non-zero exit status 1.During handling of the above exception, another exception occurred:
Traceback (most recent call last):
File "/user/HS501/fa00899/miniconda3/envs/qiime2-2022.11/lib/python3.8/site-packages/q2cli/commands.py", line 352, in call
results = action(**arguments)
File "", line 2, in denoise_single
File "/user/HS501/fa00899/miniconda3/envs/qiime2-2022.11/lib/python3.8/site-packages/qiime2/sdk/action.py", line 234, in bound_callable
outputs = self.callable_executor(scope, callable_args,
File "/user/HS501/fa00899/miniconda3/envs/qiime2-2022.11/lib/python3.8/site-packages/qiime2/sdk/action.py", line 381, in callable_executor
output_views = self._callable(**view_args)
File "/user/HS501/fa00899/miniconda3/envs/qiime2-2022.11/lib/python3.8/site-packages/q2_dada2/_denoise.py", line 244, in denoise_single
return _denoise_single(
File "/user/HS501/fa00899/miniconda3/envs/qiime2-2022.11/lib/python3.8/site-packages/q2_dada2/_denoise.py", line 229, 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.
What can I do to fix this? I assume my reads are too short? but I haven't truncated them cos it set it to 0
Thanks
How long are your reads/what primers are you using? It sounds like the reads may not be long enough to get enough overlap to merge. If this turns out to not be the issue I will have to chat with some of the other developers about what is going on and get back to you about it.