Problem running DADA2

Hi everyone,
I have some problems running DADA2. I was working with QIIME2-2018.4 and got the following error:
An error was encountered while running DADA2 in R (return code 1), please inspect stdout and stderr to learn more

I see others got the same so I uninstall QIIME2-2018.4 and install QIIME2-2018.6. However, I got the same error…

I thought it could be something with of my new sequences and I run DADA2 with previous ones that actually worked, but I got the same error, and that’s why I try uninstalling and reinstalling QIIME2

The command I used was:

qiime dada2 denoise-paired --i-demultiplexed-seqs seq-seguimiento-prueba.qza --p-trim-left-f 0 --p-trim-left-r 0 --p-trunc-len-f 275 --p-trunc-len-r 250 --o-table table-prueba1.qza --o-representative-sequences seq-prueba1.qza --o-denoising-stats denoising-stats-prueba1.qza

And I got this:
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 /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmp0471habt/forward /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmp0471habt/reverse /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmp0471habt/output.tsv.biom /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmp0471habt/track.tsv /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmp0471habt/filt_f /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmp0471habt/filt_r 275 250 0 0 2.0 2 consensus 1.0 1 1000000

Durante la inicializaci?n - Warning messages:
1: Setting LC_COLLATE failed, using “C”
2: Setting LC_TIME failed, using “C”
3: Setting LC_MESSAGES failed, using “C”
4: Setting LC_MONETARY failed, using “C”
R version 3.4.1 (2017-06-30)
Loading required package: Rcpp
DADA2 R package version: 1.6.0

  1. Filtering Error in (function (fn, fout, maxN = c(0, 0), truncQ = c(2, 2), truncLen = c(0, :
    Mismatched forward and reverse sequence files: 100000, 11102.
    Ejecuci?n interrumpida
    Traceback (most recent call last):
    File “/anaconda3/envs/qiime2-2018.6/lib/python3.5/site-packages/q2_dada2/_denoise.py”, line 229, in denoise_paired
    run_commands([cmd])
    File “/anaconda3/envs/qiime2-2018.6/lib/python3.5/site-packages/q2_dada2/_denoise.py”, line 36, in run_commands
    subprocess.run(cmd, check=True)
    File “/anaconda3/envs/qiime2-2018.6/lib/python3.5/subprocess.py”, line 398, in run
    output=stdout, stderr=stderr)
    subprocess.CalledProcessError: Command ‘[‘run_dada_paired.R’, ‘/var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmp0471habt/forward’, ‘/var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmp0471habt/reverse’, ‘/var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmp0471habt/output.tsv.biom’, ‘/var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmp0471habt/track.tsv’, ‘/var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmp0471habt/filt_f’, ‘/var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmp0471habt/filt_r’, ‘275’, ‘250’, ‘0’, ‘0’, ‘2.0’, ‘2’, ‘consensus’, ‘1.0’, ‘1’, ‘1000000’]’ returned non-zero exit status 1

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
File “/anaconda3/envs/qiime2-2018.6/lib/python3.5/site-packages/q2cli/commands.py”, line 274, in call
results = action(**arguments)
File “”, line 2, in denoise_paired
File “/anaconda3/envs/qiime2-2018.6/lib/python3.5/site-packages/qiime2/sdk/action.py”, line 232, in bound_callable
output_types, provenance)
File “/anaconda3/envs/qiime2-2018.6/lib/python3.5/site-packages/qiime2/sdk/action.py”, line 367, in callable_executor
output_views = self._callable(**view_args)
File “/anaconda3/envs/qiime2-2018.6/lib/python3.5/site-packages/q2_dada2/_denoise.py”, line 244, 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.

Any idea what I am doing wrong?

Thank you so much

Hi Danni,

I am not part of the QIIME2 team, but do have something you could check. Is there any chance that your forward and reverse reads in the demultiplexed files are in different orders? Or that there were reads removed from either the forward or reverse files that remained in the mate pair file?

2 Likes

Hi Willowblade,

I am not sure about that, however I do the same with previous runs that were ok and the same error prompt to me. So I don`t think is something with the sequences.

Hey there @Danni!

As @willowblade mentioned, you have mismatched files here:

Your reverse reads appear to have ~88k fewer reads than the forward reads. This could be caused by an incomplete download from your sequencing center. Take a look at re-obtaining your source data again, and maybe chat with your seq center for more details.

1 Like

Hi @thermokarst,

I will look for that… but how can this error still appear in previous runs that were working fine with DADA2? I mean, I thought that some new sequences have some errors so I use previous runs that really worked and the error still appearing

For the same reasons I stated above - you might have not completely transferred your data.

Thank you very much @thermokarst

I will re-download the sequences and let you know what happened…

1 Like

Hi thermokarst,

I still have some problems using DADA2. I have re-download my sequences and I double check forward and reverse reads. I found 2 mismatched files and I already corrected them. I run DADA2 again and after two days I got the same error. What I see know is something like this:

qiime dada2 denoise-paired --i-demultiplexed-seqs sec-Prep-Oaxaca2018.qza --p-trim-left-f 0 --p-trim-left-r 0 --p-trunc-len-f 275 --p-trunc-len-r 250 --o-table table-dada2-prep-oaxaca2018.qza --o-representative-sequences sec-repres-dada2-prep-oaxaca2018.qza --o-denoising-stats stats-dada2-prep-oaxaca2018.qza --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 /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/forward /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/reverse /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/output.tsv.biom /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/track.tsv /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_f /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_r 275 250 0 0 2.0 2 consensus 1.0 1 1000000

Durante la inicializaci?n - Warning messages:

1: Setting LC_COLLATE failed, using “C”

2: Setting LC_TIME failed, using “C”

3: Setting LC_MESSAGES failed, using “C”

4: Setting LC_MONETARY failed, using “C”

R version 3.4.1 (2017-06-30)

Loading required package: Rcpp

DADA2 R package version: 1.6.0

  1. Filtering The filter removed all reads: /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_f/MSM0011-3T_S7_L001_R1_001.fastq.gz and /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_r/MSM0011-3T_S7_L001_R2_001.fastq.gz not written.

The filter removed all reads: /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_f/MSM004-3T_S5_L001_R1_001.fastq.gz and /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_r/MSM004-3T_S5_L001_R2_001.fastq.gz not written.

The filter removed all reads: /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_f/MSM004P3T_S27_L001_R1_001.fastq.gz and /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_r/MSM004P3T_S27_L001_R2_001.fastq.gz not written.

The filter removed all reads: /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_f/MSM007-3T_S6_L001_R1_001.fastq.gz and /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_r/MSM007-3T_S6_L001_R2_001.fastq.gz not written.

The filter removed all reads: /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_f/MSM009H3T_S28_L001_R1_001.fastq.gz and /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_r/MSM009H3T_S28_L001_R2_001.fastq.gz not written.

The filter removed all reads: /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_f/MSM013-3T_S8_L001_R1_001.fastq.gz and /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_r/MSM013-3T_S8_L001_R2_001.fastq.gz not written.

The filter removed all reads: /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_f/MSM013H3T_S31_L001_R1_001.fastq.gz and /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_r/MSM013H3T_S31_L001_R2_001.fastq.gz not written.

The filter removed all reads: /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_f/MSM013_S42_L001_R1_001.fastq.gz and /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_r/MSM013_S42_L001_R2_001.fastq.gz not written.

The filter removed all reads: /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_f/MSM019-3T_S9_L001_R1_001.fastq.gz and /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_r/MSM019-3T_S9_L001_R2_001.fastq.gz not written.

The filter removed all reads: /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_f/MSM021-3T_S10_L001_R1_001.fastq.gz and /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_r/MSM021-3T_S10_L001_R2_001.fastq.gz not written.

The filter removed all reads: /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_f/MSM021H3T_S34_L001_R1_001.fastq.gz and /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_r/MSM021H3T_S34_L001_R2_001.fastq.gz not written.

The filter removed all reads: /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_f/MSM028-3T_S11_L001_R1_001.fastq.gz and /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_r/MSM028-3T_S11_L001_R2_001.fastq.gz not written.

The filter removed all reads: /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_f/MSM028H3T_S35_L001_R1_001.fastq.gz and /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_r/MSM028H3T_S35_L001_R2_001.fastq.gz not written.

The filter removed all reads: /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_f/MSM037_S13_L001_R1_001.fastq.gz and /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_r/MSM037_S13_L001_R2_001.fastq.gz not written.

The filter removed all reads: /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_f/MSM039_S15_L001_R1_001.fastq.gz and /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_r/MSM039_S15_L001_R2_001.fastq.gz not written.

The filter removed all reads: /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_f/MSM040_S16_L001_R1_001.fastq.gz and /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_r/MSM040_S16_L001_R2_001.fastq.gz not written.

The filter removed all reads: /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_f/MSM044_S19_L001_R1_001.fastq.gz and /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_r/MSM044_S19_L001_R2_001.fastq.gz not written.

The filter removed all reads: /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_f/MSM045_S20_L001_R1_001.fastq.gz and /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_r/MSM045_S20_L001_R2_001.fastq.gz not written.

The filter removed all reads: /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_f/MSM046_S21_L001_R1_001.fastq.gz and /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_r/MSM046_S21_L001_R2_001.fastq.gz not written.

The filter removed all reads: /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_f/MSM049_S24_L001_R1_001.fastq.gz and /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_r/MSM049_S24_L001_R2_001.fastq.gz not written.

The filter removed all reads: /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_f/MSM050_S25_L001_R1_001.fastq.gz and /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpaqv4wrz7/filt_r/MSM050_S25_L001_R2_001.fastq.gz not written.

It is still working with verbose so I don’t know if there is another problem. However, do you know why my seqs are not passing the filter?

Thank you in advance

Hmm, looks like at least one of the settings being provided to DADA2 is causing a majority of your reads to be discarded. Can you run demux summarize and provide the resulting QZV here (or DM to me if you can’t make these data public). Thanks!

Thanks for sharing your summarize viz, @Danni!

I suspect your trim and trunc params aren’t quite stringent enough. Here is where I would start, if this was my analysis (shooting for ~Q25 cutoff).

qiime dada2 denoise-paired \
  --i-demultiplexed-seqs sec-Prep-Oaxaca2018.qza \
  --p-trim-left-f 5 \
  --p-trim-left-r 5 \
  --p-trunc-len-f 253 \
  --p-trunc-len-r 225 \
  --o-table table-dada2-prep-oaxaca2018.qza \
  --o-representative-sequences sec-repres-dada2-prep-oaxaca2018.qza \
  --o-denoising-stats stats-dada2-prep-oaxaca2018.qza \
  --verbose

Also, just to confirm - have all adapter, linkers, and barcodes been removed? If not, you will need to start there prior to DADA2.

Thank you very much @thermokarst… I am going to try it and let you know what happened

1 Like

Hi,

It seems it is happening the same

(qiime2-2018.6) iMac-de-CIENI-2:Oaxaca_2018 danielagarrido$ qiime dada2 denoise-paired --i-demultiplexed-seqs sec-Prep-Oaxaca2018.qza --p-trim-left-f 5 --p-trim-left-r 5 --p-trunc-len-f 253 --p-trunc-len-r 225 --o-table table-dada2-prep-oaxaca2018.qza --o-representative-sequences sec-repres-dada2-prep-oaxaca2018.qza --o-denoising-stats stats-dada2-prep-oaxaca2018.qza --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 /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpe1wqmc9a/forward /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpe1wqmc9a/reverse /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpe1wqmc9a/output.tsv.biom /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpe1wqmc9a/track.tsv /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpe1wqmc9a/filt_f /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpe1wqmc9a/filt_r 253 225 5 5 2.0 2 consensus 1.0 1 1000000

Durante la inicializaci?n - Warning messages:

1: Setting LC_COLLATE failed, using “C”

2: Setting LC_TIME failed, using “C”

3: Setting LC_MESSAGES failed, using “C”

4: Setting LC_MONETARY failed, using “C”

R version 3.4.1 (2017-06-30)

Loading required package: Rcpp

DADA2 R package version: 1.6.0

  1. Filtering The filter removed all reads: /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpe1wqmc9a/filt_f/MSM0011-3T_S7_L001_R1_001.fastq.gz and /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpe1wqmc9a/filt_r/MSM0011-3T_S7_L001_R2_001.fastq.gz not written.

The filter removed all reads: /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpe1wqmc9a/filt_f/MSM004-3T_S5_L001_R1_001.fastq.gz and /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpe1wqmc9a/filt_r/MSM004-3T_S5_L001_R2_001.fastq.gz not written.

The filter removed all reads: /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpe1wqmc9a/filt_f/MSM004P3T_S27_L001_R1_001.fastq.gz and /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpe1wqmc9a/filt_r/MSM004P3T_S27_L001_R2_001.fastq.gz not written.

The filter removed all reads: /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpe1wqmc9a/filt_f/MSM007-3T_S6_L001_R1_001.fastq.gz and /var/folders/kp/ly6vb8yd3m55sypghf0vmblm0000gp/T/tmpe1wqmc9a/filt_r/MSM007-3T_S6_L001_R2_001.fastq.gz not written.

It is a little bit frustrating… =(

Did you see my question above?

Non-biological signal in your reads could certainly cause the issues you are seeing.

Maybe just as a sanity check you could try processing just your forward reads, and see if you see this same aggressive filtering. No need to reimport, just pass your pe reads to qiime dada2 denoise-single and it will use the forward reads.

Otherwise, I think now might be a good time to familiarize yourself with the dada2 docs, if you haven’t already reviewed them — sounds like you might need to do some additional parameter tweaking for these data. Keep us posted.

Hi,

About this…

Everything was removed

I am going to try it…

Thank you very much

1 Like

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