Dada2 to identify sequence variants with ITS data

Hi

I need help with the command for dada2 of an ITS data. After trimming the paired-end sequences with itsexpress, I encountered this error:

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 + 12 nucleotides (the length of the overlap). Alternatively, other arguments (such as max_ee or trunc_q) may be preventing reads from passing the filter.

Debug info has been saved to /tmp/qiime2-q2cli-err-xxd57hwb.log

Error log:

GNU nano 4.8 /tmp/qiime2-q2cli-err-gw2qi0do.log
Running external command-line application(s). This may print messages to stdou>
The command(s) being run are below. These commands cannot be manually re-run a>

Command: run_dada_paired.R /tmp/tmpu52d3lx0/forward /tmp/tmpu52d3lx0/reverse />

R version 4.0.2 (2020-06-22)
Loading required package: Rcpp
DADA2: 1.18.0 / Rcpp: 1.0.5 / RcppParallel: 5.0.2

  1. Filtering The filter removed all reads: /tmp/tmpu52d3lx0/filt_f/101A_8_L001>
    The filter removed all reads: /tmp/tmpu52d3lx0/filt_f/101B_6_L001_R1_001.fastq>
    The filter removed all reads: /tmp/tmpu52d3lx0/filt_f/101C_9_L001_R1_001.fastq>
    The filter removed all reads: /tmp/tmpu52d3lx0/filt_f/202A_1_L001_R1_001.fastq>
    The filter removed all reads: /tmp/tmpu52d3lx0/filt_f/202B_11_L001_R1_001.fast>
    The filter removed all reads: /tmp/tmpu52d3lx0/filt_f/202C_4_L001_R1_001.fastq>
    The filter removed all reads: /tmp/tmpu52d3lx0/filt_f/303A_7_L001_R1_001.fastq>
    The filter removed all reads: /tmp/tmpu52d3lx0/filt_f/303B_12_L001_R1_001.fast>
    The filter removed all reads: /tmp/tmpu52d3lx0/filt_f/303C_3_L001_R1_001.fastq>
    The filter removed all reads: /tmp/tmpu52d3lx0/filt_f/404A_2_L001_R1_001.fastq>
    The filter removed all reads: /tmp/tmpu52d3lx0/filt_f/404B_10_L001_R1_001.fast>
    The filter removed all reads: /tmp/tmpu52d3lx0/filt_f/404C_5_L001_R1_001.fastq>
    xxxxxxxxxxxx
    Error: No reads passed the filter (were truncLenF/R longer than the read lengt>
    Traceback (most recent call last):
    File "/home/qiime2/miniconda/envs/qiime2-2020.11/lib/python3.6/site-packages>
    run_commands([cmd])

Can anyone help with this type of error?
Regards.

Hi,
How many reads made it through the itsxpress step? (Is the issue in dada2 or itsxpress)

What was the exact dada2 command you ran?

What is the length of your reads?

1 Like

Hi Devin

The result of demux file:

Demultiplexed sequence counts summary

forward reads reverse reads
Minimum 85582 85582
Median 247188 247188
Mean 215532 215532
Maximum 328415 328415
Total 2586389 2586389

Forward Reads Frequency Histogram


Download as PDF

Reverse Reads Frequency Histogram


Download as PDF

Per-sample sequence counts

Total Samples: 12 (forward) 12 (reverse)

forward sequence count reverse sequence count
sample ID
202C 328415 328415
303C 311286 311286
404C 292870 292870
303A 285828 285828
404B 278152 278152
202A 267511 267511
303B 226865 226865
404A 165708 165708
101C 122476 122476
101B 111337 111337
101A 110359 110359
202B 85582 85582

The dada2 command:

qiime dada2 denoise-paired \

--i-demultiplexed-seqs trimmed.qza
--p-trunc-len-r 0
--p-trunc-len-f 0
--output-dir dada2out

The length of my read is 8nt.

Regards.

I would guess this may be the issue unless that's a typo, maybe related to this part.

or trunc_len_f + trunc_len_r may be shorter than the length of the amplicon + 12 nucleotides

Hi Devin
Thanks for your response.

What do you suggest I do?

Should I just use the forward reads alone?
What is the advantage of using deblur for ITS analysis (I have paired-end reads from MRDNA facility)?
Can I use it in this case?

Waiting for your advice.

Regards.

Hi @bettya!

Can you please clear up @dwt's question about your read length? 8nts looks like a typo, but if you do have reads that are only 8nts then something went wrong prior to importing your data into QIIME 2.

Keep us posted!

:qiime2:

Hi
Sorry I missed the read length part.
My sequencing read length is 2 by 150bp for a paired-end read.
From my machine I have these details of my reads:

emp-paired-end-sequences.qza (before multiplexing) - 381,388KB
demux-full.qza - 331,977KB

After trimming with itsxpress:

Trimmed.qza - 23KB
Trimmed_exact.qza - 23KB

I don't know why all the reads were reduced so much.
I realised from my demux-details.qza that my reads look trimmed and neat.
What is the implication of that? (Someone told me that sequencing reads from Illumina facility/MRDNA are quality trimmed and they are not so good with dada2 but that I should use deblur, I haven't tried that before)
Could this be the reason for the above error?

Thanks for your help.

Hi @bettya , sorry for the slow reply. It looks like your attachments didn't upload, can you please try again, thanks!

Hi thermokarst
The above are the details of my ITS sequences.

Thanks.

Hi @bettya! There was an issue uploading:

Please send the attachments again if you would like us to review them, thanks!

Hi thermokarst

Please, what do you mean by attachments?
Do you mean part of my reads/sequences?

Kindly inform.

Regards.