Please help me！I use DADA2 to generate ASV table error, request your help!

[JayZ]

qiime dada2 denoise-paired
--i-demultiplexed-seqs ./导入的序列/paired-end-demux.qza
--p-trim-left-f 23
--p-trim-left-r 23
--p-trunc-len-f 250
--p-trunc-len-r 250
--o-table ./质控聚类/table.qza
--o-representative-sequences ./质控聚类/rep-seqs.qza
--o-denoising-stats ./质控聚类/denoising-stats.qza
--p-n-threads 0
--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 /tmp/tmpq32ozyap/forward /tmp/tmpq32ozyap/reverse /tmp/tmpq32ozyap/output.tsv.biom /tmp/tmpq32ozyap/track.tsv /tmp/tmpq32ozyap/filt_f /tmp/tmpq32ozyap/filt_r 250 250 23 23 2.0 2.0 2 12 independent consensus 1.0 0 1000000

R version 4.0.5 (2021-03-31) 
Loading required package: Rcpp
DADA2: 1.18.0 / Rcpp: 1.0.7 / RcppParallel: 5.1.4 
1) Filtering Error in filterAndTrim(unfiltsF, filtsF, unfiltsR, filtsR, truncLen = c(truncLenF,  : 
  These are the errors (up to 5) encountered in individual cores...
Error in (function (fn, fout, maxN = c(0, 0), truncQ = c(2, 2), truncLen = c(0,  : 
  Mismatched forward and reverse sequence files: 51326, 60771.
Error in (function (fn, fout, maxN = c(0, 0), truncQ = c(2, 2), truncLen = c(0,  : 
  Mismatched forward and reverse sequence files: 51326, 60771.
Error in (function (fn, fout, maxN = c(0, 0), truncQ = c(2, 2), truncLen = c(0,  : 
  Mismatched forward and reverse sequence files: 51326, 60771.
Error in (function (fn, fout, maxN = c(0, 0), truncQ = c(2, 2), truncLen = c(0,  : 
  Mismatched forward and reverse sequence files: 51326, 60771.
Error in (function (fn, fout, maxN = c(0, 0), truncQ = c(2, 2), truncLen = c(0,  : 
  Mismatched forward and reverse sequence files: 51326, 60771.
Execution halted
Traceback (most recent call last):
  File "/home/qiime2/miniconda/envs/qiime2-2021.11/lib/python3.8/site-packages/q2_dada2/_denoise.py", line 266, in denoise_paired
    run_commands([cmd])
  File "/home/qiime2/miniconda/envs/qiime2-2021.11/lib/python3.8/site-packages/q2_dada2/_denoise.py", line 36, in run_commands
    subprocess.run(cmd, check=True)
  File "/home/qiime2/miniconda/envs/qiime2-2021.11/lib/python3.8/subprocess.py", line 516, in run
    raise CalledProcessError(retcode, process.args,
subprocess.CalledProcessError: Command '['run_dada_paired.R', '/tmp/tmpq32ozyap/forward', '/tmp/tmpq32ozyap/reverse', '/tmp/tmpq32ozyap/output.tsv.biom', '/tmp/tmpq32ozyap/track.tsv', '/tmp/tmpq32ozyap/filt_f', '/tmp/tmpq32ozyap/filt_r', '250', '250', '23', '23', '2.0', '2.0', '2', '12', 'independent', 'consensus', '1.0', '0', '1000000']' returned non-zero exit status 1.

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
  File "/home/qiime2/miniconda/envs/qiime2-2021.11/lib/python3.8/site-packages/q2cli/commands.py", line 339, in __call__
    results = action(**arguments)
  File "<decorator-gen-398>", line 2, in denoise_paired
  File "/home/qiime2/miniconda/envs/qiime2-2021.11/lib/python3.8/site-packages/qiime2/sdk/action.py", line 245, in bound_callable
    outputs = self._callable_executor_(scope, callable_args,
  File "/home/qiime2/miniconda/envs/qiime2-2021.11/lib/python3.8/site-packages/qiime2/sdk/action.py", line 391, in _callable_executor_
    output_views = self._callable(**view_args)
  File "/home/qiime2/miniconda/envs/qiime2-2021.11/lib/python3.8/site-packages/q2_dada2/_denoise.py", line 279, 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.

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.

[lizgehret]

Hi @JayZ,

Welcome to the :qiime2: forum!

@JayZ

Mismatched forward and reverse sequence files: 51326, 60771.

This is the the issue - which could be caused by a few different things:

• Having a differing number sequence counts between your forward and reverse reads.
• The sequences in one of the two paired files could be out of order with respect to the other.
• There could be non standard headers in one (or both) of the files that makes the parser "think" the files are mismatched.

This is actually not an uncommon issue - I've linked a few forum posts below, as well as a Github issue that will provide you with a handful of troubleshooting steps you can take to determine the source of this mismatch and how to resolve it.

Mismatched forward and reverse reads

Mismatched forward and reverse sequence files - files don't exist

Hello, I am currently using qiime2-2019.10. While running DADA2 I ran into a mismatched forward and reverse sequence file error. I found similar issues in other threads in the forum and attempted to repeat the steps but the mismatched files don’t seem to exist. My input and the qiime2 output is below: qiime dada2 denoise-paired \ > --i-demultiplexed-seqs demux-paired-end.qza \ > --p-trim-left-f 13 \ > --p-trim-left-r 13 \ > --p-trunc-len-f 247 \ > --p-trunc-len-r 247 \ > --…

Mismatched forward and reverse sequence files

hello I have a problem with DADA2 filtering The output result seems there is a mismatch between my forward and reverse file I have already checked similar issues on the forum https://forum.qiime2.org/t/error-in-filtering-using-dada2/3543/11 https://forum.qiime2.org/t/mismatched-forward-and-reverse-sequence-files-files-dont-exist/12511 here are my input and output #input #import data qiime tools import \ --type 'SampleData[PairedEndSequencesWithQuality]' \ --input-path /home/chaojuic…

Hope this helps!

Cheers