Hi everyone, Sorry to bothered your gays. I am a beginner of QIIME2, which is truly confused me a lot. So I want ask for help in the forum.
This is a problem I encountered when I used dada2 to generate a features table. The following is my procedures:
- I download a paired-end 16S data from NCBI in the form of .fastq(include two files, one is forward, the other is reverse)
2.I used fastp to trim adapters and filter out low-quality sequences from the paired-end 16S rRNA gene sequencing files.
3.I import them into qiime2 with the following code:
time qiime tools import \
--type 'SampleData[PairedEndSequencesWithQuality]' \
--input-path /mypath/SRR19603331/manifeat.tsv \
--output-path paired-end-demux_1.qza \
--input-format PairedEndFastqManifestPhred33V2
- I wanted to use dada2 to generated a feature table by following code:
qiime dada2 denoise-paired \
--i-demultiplexed-seqs paired-end-demux_1.qza \
--p-trunc-len-f 0 \
--p-trunc-len-r 0 \
--p-n-threads 20 \
--o-table table.qza \
--o-representative-sequences rep-seqs.qza \
--o-denoising-stats denoising-stats.qza
However, it encountered a error and return as following:
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-1xrwtbtm.log
Thank you all in advance for your help and support. I truly appreciate any guidance you can provide.
The Debug info file is as following:
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/tmp22ui1r15/forward --input_directory_reverse /tmp/tmp22ui1r15/reverse --output_path /tmp/tmp22ui1r15/output.tsv.biom --output_track /tmp/tmp22ui1r15/track.tsv --filtered_directory /tmp/tmp22ui1r15/filt_f --filtered_directory_reverse /tmp/tmp22ui1r15/filt_r --truncation_length 0 --truncation_length_reverse 0 --trim_left 0 --trim_left_reverse 0 --max_expected_errors 2.0 --max_expected_errors_reverse 2.0 --truncation_quality_score 2 --min_overlap 12 --pooling_method independent --chimera_method consensus --min_parental_fold 1.0 --allow_one_off False --num_threads 20 --learn_min_reads 1000000
R version 4.3.3 (2024-02-29)
Loading required package: Rcpp
DADA2: 1.30.0 / Rcpp: 1.0.12 / RcppParallel: 5.1.6
2) Filtering .
3) Learning Error Rates
44549066 total bases in 177490 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)
2: withCallingHandlers(expr, warning = function(w) if (inherits(w,
classes)) tryInvokeRestart("muffleWarning"))
1: suppressWarnings(learnErrors(filts, nreads = nreads.learn, multithread = multithread))
Traceback (most recent call last):
File "/home/zcw/miniconda3/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/q2_dada2/_denoise.py", line 350, in denoise_paired
run_commands([cmd])
File "/home/zcw/miniconda3/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/q2_dada2/_denoise.py", line 37, in run_commands
subprocess.run(cmd, check=True)
File "/home/zcw/miniconda3/envs/qiime2-amplicon-2024.5/lib/python3.9/subprocess.py", line 528, in run
raise CalledProcessError(retcode, process.args,
subprocess.CalledProcessError: Command '['run_dada.R', '--input_directory', '/tmp/tmp22ui1r15/forward', '--input_directory_reverse', '/tmp/tmp22ui1r15/reverse', '--output_path', '/tmp/tmp22ui1r15/output.tsv.biom', '--output_track', '/tmp/tmp22ui1r15/track.tsv', '--filtered_directory', '/tmp/tmp22ui1r15/filt_f', '--filtered_directory_reverse', '/tmp/tmp22ui1r15/filt_r', '--truncation_length', '0', '--truncation_length_reverse', '0', '--trim_left', '0', '--trim_left_reverse', '0', '--max_expected_errors', '2.0', '--max_expected_errors_reverse', '2.0', '--truncation_quality_score', '2', '--min_overlap', '12', '--pooling_method', 'independent', '--chimera_method', 'consensus', '--min_parental_fold', '1.0', '--allow_one_off', 'False', '--num_threads', '20', '--learn_min_reads', '1000000']' returned non-zero exit status 1.
During handling of the above exception, another exception occurred:
Traceback (most recent call last):
File "/home/zcw/miniconda3/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/q2cli/commands.py", line 520, in call
results = self._execute_action(
File "/home/zcw/miniconda3/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/q2cli/commands.py", line 581, in _execute_action
results = action(**arguments)
File "", line 2, in denoise_paired
File "/home/zcw/miniconda3/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/qiime2/sdk/action.py", line 342, in bound_callable
outputs = self.callable_executor(
File "/home/zcw/miniconda3/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/qiime2/sdk/action.py", line 576, in callable_executor
output_views = self._callable(**view_args)
File "/home/zcw/miniconda3/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/q2_dada2/_denoise.py", line 363, 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.