Error when using DAD2 denosie with qiime2-2019.1 (the error file content included)

Hi,
I have only one sample sequenced and the read count is too low (272 reads found in demux. qzv file). when I run dada2 plugin:
qiime dada2 denoise-paired --i-demultiplexed-seqs demux-paired-end.qza --p-trunc-len-f 245 --p-trunc-len-r 245 --p-trim-left-f 19 --p-trim-left-r 20 --p-n-threads 3 --p-n-reads-learn 1000000 --output-dir representative_seq --verbose

I got this error: (need to understand, what does it mean and what should I do). Thanks

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/tmpmz0_hq01/forward /tmp/tmpmz0_hq01/reverse /tmp/tmpmz0_hq01/output.tsv.biom /tmp/tmpmz0_hq01/track.tsv /tmp/tmpmz0_hq01/filt_f /tmp/tmpmz0_hq01/filt_r 245 245 19 20 2.0 2 consensus 1.0 3 1000000

R version 3.4.1 (2017-06-30)
Loading required package: Rcpp
DADA2 R package version: 1.6.0

  1. Filtering .

  2. Learning Error Rates
    2a) Forward Reads
    Initializing error rates to maximum possible estimate.
    Sample 1 - 230 reads in 84 unique sequences.
    selfConsist step 2
    Convergence after 2 rounds.
    2b) Reverse Reads
    Initializing error rates to maximum possible estimate.
    Sample 1 - 230 reads in 108 unique sequences.
    selfConsist step 2
    Convergence after 2 rounds.

  3. Denoise remaining samples

  4. Remove chimeras (method = consensus)
    Error in isBimeraDenovoTable(unqs[[i]], …, verbose = verbose) :
    Input must be a valid sequence table.
    Calls: removeBimeraDenovo -> isBimeraDenovoTable
    In addition: Warning message:
    In is.na(colnames(unqs[[i]])) :
    is.na() applied to non-(list or vector) of type β€˜NULL’
    Execution halted
    Traceback (most recent call last):
    File β€œ/home/eman/anaconda3/envs/qiime2-2019.1/lib/python3.6/site-packages/q2_dada2/_denoise.py”, line 231, in denoise_paired
    run_commands([cmd])
    File β€œ/home/eman/anaconda3/envs/qiime2-2019.1/lib/python3.6/site-packages/q2_dada2/_denoise.py”, line 36, in run_commands
    subprocess.run(cmd, check=True)
    File β€œ/home/eman/anaconda3/envs/qiime2-2019.1/lib/python3.6/subprocess.py”, line 418, in run
    output=stdout, stderr=stderr)
    subprocess.CalledProcessError: Command β€˜[β€˜run_dada_paired.R’, β€˜/tmp/tmpmz0_hq01/forward’, β€˜/tmp/tmpmz0_hq01/reverse’, β€˜/tmp/tmpmz0_hq01/output.tsv.biom’, β€˜/tmp/tmpmz0_hq01/track.tsv’, β€˜/tmp/tmpmz0_hq01/filt_f’, β€˜/tmp/tmpmz0_hq01/filt_r’, β€˜245’, β€˜245’, β€˜19’, β€˜20’, β€˜2.0’, β€˜2’, β€˜consensus’, β€˜1.0’, β€˜3’, β€˜1000000’]’ returned non-zero exit status 1.

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
File β€œ/home/eman/anaconda3/envs/qiime2-2019.1/lib/python3.6/site-packages/q2cli/commands.py”, line 274, in call
results = action(**arguments)
File β€œ</home/eman/anaconda3/envs/qiime2-2019.1/lib/python3.6/site-packages/decorator.py:decorator-gen-442>”, line 2, in denoise_paired
File β€œ/home/eman/anaconda3/envs/qiime2-2019.1/lib/python3.6/site-packages/qiime2/sdk/action.py”, line 231, in bound_callable
output_types, provenance)
File β€œ/home/eman/anaconda3/envs/qiime2-2019.1/lib/python3.6/site-packages/qiime2/sdk/action.py”, line 365, in callable_executor
output_views = self._callable(**view_args)
File β€œ/home/eman/anaconda3/envs/qiime2-2019.1/lib/python3.6/site-packages/q2_dada2/_denoise.py”, line 246, in denoise_paired
" and stderr to learn more." % e.returncode)
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.

Hello @Eman!

Yikes! You won't be able to do much with one sample besides taxonomic classification --- diversity stats are out of the picture...

Check out this part of the error message:

This means that, after trim/truncation, DADA2 was unable to join your forward and reverse reads. DADA2 needs at least 20 nt overlap in order to join reads. Options include adjusting your trim/trunc params to see if you can make it work, or proceeding with just the forward (or reverse) reads. Either way, with only one sample and 272 reads, there isn't too much you can do with these data. Keep us posted! :t_rex:

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Does it mean the quality of sequences is poor?

I got the same message (Error in isBimeraDenovoTable(…) when run DADA denoise on 4 samples (to detect 16S in fungal DNA), so that means there is no enough 16S to be amplified? It is a sequencing test to see whether to sequence the rest of the samples or not?

No, it means that the reads were not overlapping enough for DADA2 to join them.

But I am using the maximum possible length. Do you mind sharing demux.qzv files? just take a look and let me know if I did aggressive trimming or not. I did the same in previous projects and it was okay!!

Perhaps this is a wet-lab or sequencing issue, then? Maybe something is wrong with the primers used?

Feel free to send them!

demux-paired-end.qzv (273.4 KB)demux-paired-end.qzv (293.5 KB)

Thanks for your patience

There are so few samples and reads in both of these summaries, I think you might have a hard time conducting any type of meaningful analysis here. The summary with 4 samples has very low quality reads in the forward direction, which is probably going to cause an issue with read joining. The parameters you posted above seem reasonable to me, given the summaries. What did the sequencing center have to say about the run? Any thoughts on why you have such low sequencing coverage?

Yes, I sent them an inquiry about this issue, especially I have other samples that I could generate representative _seq, but they are almost empty from 16S, just have chloroplast sequences (my DNA samples from plant tissues). However, I had good sequences from similar tissues last year, but they were using primers for v3-v4 region.
This time, they are using different primers set, but still amplifying part of 16S (v4-v5). So, what we recommended is to re-sequence samples but using other previously used primers (v4 region), that we recently used in other project in different plant tissues.
Do you think changing the primers would make a difference in sequencing??
I am now waiting for the new sequences which may take 1-2 weeks.
Will keep the forum updated to know whether it is a technical issue or not?

Thank you so much.

Almost certainly.

Please do!

Hi @thermokarst,
I re-sequenced the samples but with different primers set (for v4 region), and got good reads. So the issue maybe technical and or due to primers selection.
Thanks

1 Like

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