Error in Dada2 analysis after ITS sequence trimming analysis

User Support
1

Hi, All

I have followed ITS Xpress-tutorial for my ITS1 and ITS2 sequence processing and taxonomy analysis. i got error in DADA 2 analysis after ITS samples trimming step.

(qiime2-2019.10) bioinformatics@bioinformatics-desktop:~/.conda/envs/qiime2-2019.10$ qiime dada2 denoise-paired --i-demultiplexed-seqs trimmed.qza --p-trunc-len-r 0 --p-trunc-len-f 0 --p-chimera-method none --p-trunc-q 0 --p-max-ee-r 1 --p-max-ee-f 1 --output-dir dada2out --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/tmpkfw2u9k8/forward /tmp/tmpkfw2u9k8/reverse /tmp/tmpkfw2u9k8/output.tsv.biom /tmp/tmpkfw2u9k8/track.tsv /tmp/tmpkfw2u9k8/filt_f /tmp/tmpkfw2u9k8/filt_r 0 0 0 0 1.0 1.0 0 none 1.0 1 1000000

R version 3.5.1 (2018-07-02)
Loading required package: Rcpp
DADA2: 1.10.0 / Rcpp: 1.0.2 / RcppParallel: 4.4.4

  1. Filtering The filter removed all reads: /tmp/tmpkfw2u9k8/filt_f/sample-1_0_L001_R1_001.fastq.gz and /tmp/tmpkfw2u9k8/filt_r/sample-1_1_L001_R2_001.fastq.gz not written.
    The filter removed all reads: /tmp/tmpkfw2u9k8/filt_f/sample-10_18_L001_R1_001.fastq.gz and /tmp/tmpkfw2u9k8/filt_r/sample-10_19_L001_R2_001.fastq.gz not written.
    The filter removed all reads: /tmp/tmpkfw2u9k8/filt_f/sample-11_20_L001_R1_001.fastq.gz and /tmp/tmpkfw2u9k8/filt_r/sample-11_21_L001_R2_001.fastq.gz not written.
    The filter removed all reads: /tmp/tmpkfw2u9k8/filt_f/sample-12_22_L001_R1_001.fastq.gz and /tmp/tmpkfw2u9k8/filt_r/sample-12_23_L001_R2_001.fastq.gz not written.
    The filter removed all reads: /tmp/tmpkfw2u9k8/filt_f/sample-2_2_L001_R1_001.fastq.gz and /tmp/tmpkfw2u9k8/filt_r/sample-2_3_L001_R2_001.fastq.gz not written.
    The filter removed all reads: /tmp/tmpkfw2u9k8/filt_f/sample-3_4_L001_R1_001.fastq.gz and /tmp/tmpkfw2u9k8/filt_r/sample-3_5_L001_R2_001.fastq.gz not written.
    The filter removed all reads: /tmp/tmpkfw2u9k8/filt_f/sample-4_6_L001_R1_001.fastq.gz and /tmp/tmpkfw2u9k8/filt_r/sample-4_7_L001_R2_001.fastq.gz not written.
    The filter removed all reads: /tmp/tmpkfw2u9k8/filt_f/sample-5_8_L001_R1_001.fastq.gz and /tmp/tmpkfw2u9k8/filt_r/sample-5_9_L001_R2_001.fastq.gz not written.
    The filter removed all reads: /tmp/tmpkfw2u9k8/filt_f/sample-6_10_L001_R1_001.fastq.gz and /tmp/tmpkfw2u9k8/filt_r/sample-6_11_L001_R2_001.fastq.gz not written.
    The filter removed all reads: /tmp/tmpkfw2u9k8/filt_f/sample-7_12_L001_R1_001.fastq.gz and /tmp/tmpkfw2u9k8/filt_r/sample-7_13_L001_R2_001.fastq.gz not written.
    The filter removed all reads: /tmp/tmpkfw2u9k8/filt_f/sample-8_14_L001_R1_001.fastq.gz and /tmp/tmpkfw2u9k8/filt_r/sample-8_15_L001_R2_001.fastq.gz not written.
    The filter removed all reads: /tmp/tmpkfw2u9k8/filt_f/sample-9_16_L001_R1_001.fastq.gz and /tmp/tmpkfw2u9k8/filt_r/sample-9_17_L001_R2_001.fastq.gz not written.
    xxxxxxxxxxxx
    Error: No reads passed the filter (were truncLenF/R longer than the read lengths?)
    Traceback (most recent call last):
    File "/home/bioinformatics/.conda/envs/qiime2-2019.10/lib/python3.6/site-packages/q2_dada2/_denoise.py", line 257, in denoise_paired
    run_commands([cmd])
    File "/home/bioinformatics/.conda/envs/qiime2-2019.10/lib/python3.6/site-packages/q2_dada2/_denoise.py", line 36, in run_commands
    subprocess.run(cmd, check=True)
    File "/home/bioinformatics/.conda/envs/qiime2-2019.10/lib/python3.6/subprocess.py", line 418, in run
    output=stdout, stderr=stderr)
    subprocess.CalledProcessError: Command '['run_dada_paired.R', '/tmp/tmpkfw2u9k8/forward', '/tmp/tmpkfw2u9k8/reverse', '/tmp/tmpkfw2u9k8/output.tsv.biom', '/tmp/tmpkfw2u9k8/track.tsv', '/tmp/tmpkfw2u9k8/filt_f', '/tmp/tmpkfw2u9k8/filt_r', '0', '0', '0', '0', '1.0', '1.0', '0', 'none', '1.0', '1', '1000000']' returned non-zero exit status 2.

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
File "/home/bioinformatics/.conda/envs/qiime2-2019.10/lib/python3.6/site-packages/q2cli/commands.py", line 328, in call
results = action(**arguments)
File "</home/bioinformatics/.conda/envs/qiime2-2019.10/lib/python3.6/site-packages/decorator.py:decorator-gen-465>", line 2, in denoise_paired
File "/home/bioinformatics/.conda/envs/qiime2-2019.10/lib/python3.6/site-packages/qiime2/sdk/action.py", line 240, in bound_callable
output_types, provenance)
File "/home/bioinformatics/.conda/envs/qiime2-2019.10/lib/python3.6/site-packages/qiime2/sdk/action.py", line 383, in callable_executor
output_views = self._callable(**view_args)
File "/home/bioinformatics/.conda/envs/qiime2-2019.10/lib/python3.6/site-packages/q2_dada2/_denoise.py", line 268, in denoise_paired
% (trunc_len_f, trunc_len_r))
ValueError: No reads passed the filter. trunc_len_f (0) or trunc_len_r (0) may be individually longer than read lengths, or trunc_len_f + trunc_len_r may be shorter than the length of the amplicon + 20 nucleotides (the length of the overlap). Alternatively, other arguments (such as max_ee or trunc_q) may be preventing reads from passing the filter.

Plugin error from dada2:

No reads passed the filter. trunc_len_f (0) or trunc_len_r (0) may be individually longer than read lengths, or trunc_len_f + trunc_len_r may be shorter than the length of the amplicon + 20 nucleotides (the length of the overlap). Alternatively, other arguments (such as max_ee or trunc_q) may be preventing reads from passing the filter.

See above for debug info.

Could anyone help me to solve this error ?.

Herewith, I have attached my sequence file for your perusal. kindly have a look on it.trimmed.qza (17.3 KB) sequences.qzv (290.3 KB)

2

Hi @Asha1,
No reads are passing the filter. The sequence quality is not great, so setting these parameters to zero is probably not a good idea:

You need to truncate off the low-quality 3' ends or dada2 will toss out all reads with more than a single error (since you are also setting p-max-ee-r 1 --p-max-ee-f 1)

Good luck!

3

Thank you so much for your reply and suggestion. I will cut my low quality reads as per your suggestions and get back to you soon.

1 Like
4

Hello sir,

I am getting same error continuously after I tried different phred score value. Kindly help me how to solve this error?

(qiime2-2019.10) bioinformatics@bioinformatics-desktop:~/.conda/envs/qiime2-2019.10$ qiime dada2 denoise-paired --i-demultiplexed-seqs trimmed.qza --p-trunc-len-r 296 --p-trunc-len-f 259 --output-dir dada2out --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/tmpz638ul6d/forward /tmp/tmpz638ul6d/reverse /tmp/tmpz638ul6d/output.tsv.biom /tmp/tmpz638ul6d/track.tsv /tmp/tmpz638ul6d/filt_f /tmp/tmpz638ul6d/filt_r 259 296 0 0 2.0 2.0 2 consensus 1.0 1 1000000

R version 3.5.1 (2018-07-02)
Loading required package: Rcpp
DADA2: 1.10.0 / Rcpp: 1.0.2 / RcppParallel: 4.4.4

  1. Filtering The filter removed all reads: /tmp/tmpz638ul6d/filt_f/sample-1_0_L001_R1_001.fastq.gz and /tmp/tmpz638ul6d/filt_r/sample-1_1_L001_R2_001.fastq.gz not written.
    The filter removed all reads: /tmp/tmpz638ul6d/filt_f/sample-10_18_L001_R1_001.fastq.gz and /tmp/tmpz638ul6d/filt_r/sample-10_19_L001_R2_001.fastq.gz not written.
    The filter removed all reads: /tmp/tmpz638ul6d/filt_f/sample-11_20_L001_R1_001.fastq.gz and /tmp/tmpz638ul6d/filt_r/sample-11_21_L001_R2_001.fastq.gz not written.
    The filter removed all reads: /tmp/tmpz638ul6d/filt_f/sample-12_22_L001_R1_001.fastq.gz and /tmp/tmpz638ul6d/filt_r/sample-12_23_L001_R2_001.fastq.gz not written.
    The filter removed all reads: /tmp/tmpz638ul6d/filt_f/sample-2_2_L001_R1_001.fastq.gz and /tmp/tmpz638ul6d/filt_r/sample-2_3_L001_R2_001.fastq.gz not written.
    The filter removed all reads: /tmp/tmpz638ul6d/filt_f/sample-3_4_L001_R1_001.fastq.gz and /tmp/tmpz638ul6d/filt_r/sample-3_5_L001_R2_001.fastq.gz not written.
    The filter removed all reads: /tmp/tmpz638ul6d/filt_f/sample-4_6_L001_R1_001.fastq.gz and /tmp/tmpz638ul6d/filt_r/sample-4_7_L001_R2_001.fastq.gz not written.
    The filter removed all reads: /tmp/tmpz638ul6d/filt_f/sample-5_8_L001_R1_001.fastq.gz and /tmp/tmpz638ul6d/filt_r/sample-5_9_L001_R2_001.fastq.gz not written.
    The filter removed all reads: /tmp/tmpz638ul6d/filt_f/sample-6_10_L001_R1_001.fastq.gz and /tmp/tmpz638ul6d/filt_r/sample-6_11_L001_R2_001.fastq.gz not written.
    The filter removed all reads: /tmp/tmpz638ul6d/filt_f/sample-7_12_L001_R1_001.fastq.gz and /tmp/tmpz638ul6d/filt_r/sample-7_13_L001_R2_001.fastq.gz not written.
    The filter removed all reads: /tmp/tmpz638ul6d/filt_f/sample-8_14_L001_R1_001.fastq.gz and /tmp/tmpz638ul6d/filt_r/sample-8_15_L001_R2_001.fastq.gz not written.
    The filter removed all reads: /tmp/tmpz638ul6d/filt_f/sample-9_16_L001_R1_001.fastq.gz and /tmp/tmpz638ul6d/filt_r/sample-9_17_L001_R2_001.fastq.gz not written.
    xxxxxxxxxxxx
    Error: No reads passed the filter (were truncLenF/R longer than the read lengths?)
    Traceback (most recent call last):
    File “/home/bioinformatics/.conda/envs/qiime2-2019.10/lib/python3.6/site-packages/q2_dada2/_denoise.py”, line 257, in denoise_paired
    run_commands([cmd])
    File “/home/bioinformatics/.conda/envs/qiime2-2019.10/lib/python3.6/site-packages/q2_dada2/_denoise.py”, line 36, in run_commands
    subprocess.run(cmd, check=True)
    File “/home/bioinformatics/.conda/envs/qiime2-2019.10/lib/python3.6/subprocess.py”, line 418, in run
    output=stdout, stderr=stderr)
    subprocess.CalledProcessError: Command ‘[‘run_dada_paired.R’, ‘/tmp/tmpz638ul6d/forward’, ‘/tmp/tmpz638ul6d/reverse’, ‘/tmp/tmpz638ul6d/output.tsv.biom’, ‘/tmp/tmpz638ul6d/track.tsv’, ‘/tmp/tmpz638ul6d/filt_f’, ‘/tmp/tmpz638ul6d/filt_r’, ‘259’, ‘296’, ‘0’, ‘0’, ‘2.0’, ‘2.0’, ‘2’, ‘consensus’, ‘1.0’, ‘1’, ‘1000000’]’ returned non-zero exit status 2.

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
File “/home/bioinformatics/.conda/envs/qiime2-2019.10/lib/python3.6/site-packages/q2cli/commands.py”, line 328, in call
results = action(**arguments)
File “</home/bioinformatics/.conda/envs/qiime2-2019.10/lib/python3.6/site-packages/decorator.py:decorator-gen-465>”, line 2, in denoise_paired
File “/home/bioinformatics/.conda/envs/qiime2-2019.10/lib/python3.6/site-packages/qiime2/sdk/action.py”, line 240, in bound_callable
output_types, provenance)
File “/home/bioinformatics/.conda/envs/qiime2-2019.10/lib/python3.6/site-packages/qiime2/sdk/action.py”, line 383, in callable_executor
output_views = self._callable(**view_args)
File “/home/bioinformatics/.conda/envs/qiime2-2019.10/lib/python3.6/site-packages/q2_dada2/_denoise.py”, line 268, in denoise_paired
% (trunc_len_f, trunc_len_r))
ValueError: No reads passed the filter. trunc_len_f (259) or trunc_len_r (296) may be individually longer than read lengths, or trunc_len_f + trunc_len_r may be shorter than the length of the amplicon + 20 nucleotides (the length of the overlap). Alternatively, other arguments (such as max_ee or trunc_q) may be preventing reads from passing the filter.

Plugin error from dada2:

No reads passed the filter. trunc_len_f (259) or trunc_len_r (296) may be individually longer than read lengths, or trunc_len_f + trunc_len_r may be shorter than the length of the amplicon + 20 nucleotides (the length of the overlap). Alternatively, other arguments (such as max_ee or trunc_q) may be preventing reads from passing the filter.

See above for debug info.

5

Hi sir,

I am following ITS XPRESS tutorial for my ITS1 and ITS2 sequence processing. No reads passed the filter, error was coming again and again If I give trimmed Its sequence file (trimmed.qza) for Dada2 analysis after trying different truncation value.

If I give sequence.qza for dada2 analysis file mean, I was not getting any error even with truncation value 0.

Could I use sequence.qza file for dada2 analysis without trimming the ITS sequence ?

Will it affect further processing or not?

6

I am not sure why that would be (itsxpress is only trimming to the ITS domain as far as I know, but the sequences.qzv file you shared above, which I assume is post-itsxpress, shows plenty of seqs). I am pinging @Adam_Rivers to see if he has any insight on this.

Honestly, the quality looks fairly poor on the reverse reads, though — so you may just want to proceed using only the forward reads since usually ITS reads have trouble merging (due to length variation) even when the reverse reads are good quality!

7

Ok sir. Thank you once again for your suggestions and help.

1 Like
8

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