issue with qiime dada2 denoise-ccs

I have 24 clean CCS read datasets and i want to get amplicon sequence variants by running dada2.
I got one issue when running dada2 denoise-ccs with QIIME2.

When i am running dada2 in qiime2 with all 24 samples:
The command I used was:

qiime dada2 denoise-ccs --i-demultiplexed-seqs reads_qza/ccs_reads.qza --p-min-len 1200 --p-max-len 1800 --p-front AGRGTTYGATYMTGGCTCAG --p-adapter RGYTACCTTGTTACGACTT --p-n-threads 40 --o-table dada2_output/table.qza --o-representative-sequences dada2_output/representative_sequences.qza --o-denoising-stats dada2_output/stats.qza --verbose

The debug for this error is here.

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/zcw/data/bc625311-79dd-4d22-b6e8-aefa5e8e080b/data --output_path /tmp/tmpuxwxzmu_/output.tsv.biom --output_track /tmp/tmpuxwxzmu_/track.tsv --removed_primer_directory /tmp/tmpuxwxzmu_/nop --filtered_directory /tmp/tmpuxwxzmu_/filt --forward_primer AGRGTTYGATYMTGGCTCAG --reverse_primer RGYTACCTTGTTACGACTT --max_mismatch 2 --indels False --truncation_length 0 --trim_left 0 --max_expected_errors 2.0 --truncation_quality_score 2 --min_length 1200 --max_length 1800 --pooling_method independent --chimera_method consensus --min_parental_fold 3.5 --allow_one_off False --num_threads 40 --learn_min_reads 1000000 --homopolymer_gap_penalty NULL --band_size 32

R version 4.1.3 (2022-03-10)
Loading required package: Rcpp
DADA2: 1.22.0 / Rcpp: 1.0.9 / RcppParallel: 5.1.5

Removing Primers
Read in 12234, output 0 (0%) filtered sequences.
Read in 10596, output 0 (0%) filtered sequences.
Error in sapply(match.fwd, end) + 1 :
non-numeric argument to binary operator
Execution halted
Traceback (most recent call last):
File "/home/kuma/miniconda3/envs/qiime2-2022.8/lib/python3.8/site-packages/q2_dada2/", line 410, in denoise_ccs
File "/home/kuma/miniconda3/envs/qiime2-2022.8/lib/python3.8/site-packages/q2_dada2/", line 36, in run_commands, check=True)
File "/home/kuma/miniconda3/envs/qiime2-2022.8/lib/python3.8/", line 516, in run
raise CalledProcessError(retcode, process.args,
subprocess.CalledProcessError: Command '['run_dada.R', '--input_directory', '/tmp/qiime2/zcw/data/bc625311-79dd-4d22-b6e8-aefa5e8e080b/data', '--output_path', '/tmp/tmpuxwxzmu/output.tsv.biom', '--output_track', '/tmp/tmpuxwxzmu/track.tsv', '--removed_primer_directory', '/tmp/tmpuxwxzmu_/nop', '--filtered_directory', '/tmp/tmpuxwxzmu_/filt', '--forward_primer', 'AGRGTTYGATYMTGGCTCAG', '--reverse_primer', 'RGYTACCTTGTTACGACTT', '--max_mismatch', '2', '--indels', 'False', '--truncation_length', '0', '--trim_left', '0', '--max_expected_errors', '2.0', '--truncation_quality_score', '2', '--min_length', '1200', '--max_length', '1800', '--pooling_method', 'independent', '--chimera_method', 'consensus', '--min_parental_fold', '3.5', '--allow_one_off', 'False', '--num_threads', '40', '--learn_min_reads', '1000000', '--homopolymer_gap_penalty', 'NULL', '--band_size', '32']' returned non-zero exit status 1.
During handling of the above exception, another exception occurred:

Traceback (most recent call last):
File "/home/kuma/miniconda3/envs/qiime2-2022.8/lib/python3.8/site-packages/q2cli/", line 339, in call
results = action(**arguments)
File "", line 2, in denoise_ccs
File "/home/kuma/miniconda3/envs/qiime2-2022.8/lib/python3.8/site-packages/qiime2/sdk/", line 234, in bound_callable
outputs = self.callable_executor(scope, callable_args,
File "/home/kuma/miniconda3/envs/qiime2-2022.8/lib/python3.8/site-packages/qiime2/sdk/", line 381, in callable_executor
output_views = self._callable(**view_args)
File "/home/kuma/miniconda3/envs/qiime2-2022.8/lib/python3.8/site-packages/q2_dada2/", line 419, in denoise_ccs
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.

See above for debug info

Is it possible caused by the input of the clean reads, since no primers have to be removed?
QIIME2 was installed by conda on a CentOs server.
Can you give some suggestions?

Hello @daisykuma22,

If you feel comfortable doing so, could you share your demux visualization?

clean_reads_summary.qzv (309.0 KB)
The summary of reads ia above. It seems there is nothing wrong.

Hello @daisykuma22,

The command line help text says:

Reads that do not contain the primer are discarded.

So yes that is probably the problem.

1 Like

yes, thank you. maybe i can skip primer removal.

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