Q2-ITSxpress dada2 keeps giving an error


#1

Hi,

I’m relatively new to qiime2 but familiar with 16s part. Now I wanted to make use of the q2-itsxpress. I demultiplexed my paired end reads demuxITS.qzv (293.6 KB) and then trimmed by ITSxpress with the following command:

qiime itsxpress trim-pair-output-unmerged
–i-per-sample-sequences demuxITS.qza
–p-region ITS1
–p-taxa F
–o-trimmed trimmedITS1.qza

Subsequently, I wanted to use Dada2 with this command:

qiime dada2 denoise-paired
–i-demultiplexed-seqs trimmedITS1.qza
–p-trunc-len-r 182
–p-trunc-len-f 0
–verbose
–output-dir dada2out

However, this gives the following 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 + 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.

Then I tried:

qiime dada2 denoise-paired
–i-demultiplexed-seqs trimmedITS1.qza
–o-representative-sequences rep-seqs.qza
–o-table table.qza --o-denoising-stats denoising-stats.qza
–verbose
–p-n-threads 0
–p-trunc-len-f 0
–p-trunc-len-r 182
–p-trunc-q 10

But this gave the exact same error message.
I’ve tried multiple versions of qiime2 (2018.6, 2018.11), but I’m using 2019.1 at the moment with the hope that that would help. Unfortunately for some reason I can’t get dada2 to work.

Is there

Version specs:

System versions
Python version: 3.6.5
QIIME 2 release: 2019.1
QIIME 2 version: 2019.1.0
q2cli version: 2019.1.0

Installed plugins
alignment: 2019.1.0
composition: 2019.1.0
cutadapt: 2019.1.0
dada2: 2019.1.0
deblur: 2019.1.0
demux: 2019.1.0
diversity: 2019.1.0
emperor: 2019.1.0
feature-classifier: 2019.1.0
feature-table: 2019.1.0
fragment-insertion: 2019.1.0
gneiss: 2019.1.0
longitudinal: 2019.1.0
metadata: 2019.1.0
phylogeny: 2019.1.0
quality-control: 2019.1.0
quality-filter: 2019.1.0
sample-classifier: 2019.1.0
taxa: 2019.1.0
types: 2019.1.0
vsearch: 2019.1.0

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(Matthew Ryan Dillon) #2

Hey there @persaj!

The error message is telling you that your reads aren’t being merged — with the parameters provided there is insufficient overlap to merge forward and reverse.

Let’s take a step back — do you have a demux summarize viz from after trimming your reads with itsxpress? It would be helpful to look at that, in order to contextualize your choice of truncation parameters.

Thanks! :qiime2:


#3

Hi @thermokarst!

Trying to visualize gives me another error message:

qiime demux summarize
–i-data trimmedITS1.qza
–o-visualization trimmedITS1.qzv

Cannot describe a DataFrame without columns

Thanks!


(Luca) #4

Hi @persaj,

I had the same error. I lost the original post but the problem arises from a incomplete manifest file produced by ITSxpress for the trimmed sequences. In this manifest file there is no read 2 listed.
You can see this if you use qiime2 export command on your ‘trimmedITS1.qza’. In which you may find the trimmed sequences and the wrong manifest file. I solved the issue by correcting this manifest file and importing the ITS trimmed sequences into a new qza object. This should work properly both with the ‘demux summarize’ comand and the dada2 denoising step.

Hope it makes sense.

Luca


#5

Hi @llenzi

Thanks for that, it does make sense and the manifest file was indeed incomplete. I tried to fix it, import it and then summarize it again, but it still gives me the same error. Interestingly, every file is quite small as well, for example: ‘1AL_30_L001_R1_001.fastq.gz’ is 50 bytes
I then also checked the size of the other files, my ‘demuxITS.qza’ for example is 138 MB (and emp-paired-end-sequences-ITS.qza 2.2 GB), while the ‘trimmedITS1.qza’ is 33 KB. It seems to me like a big loss of data… is that right?

Thanks!


(Luca) #6

Hi @persaj,
not sure about that, if you open the trimmed files, how many sequences are there?
It may be the case to tag @Adam_Rivers for help!
Luca


(Matthew Ryan Dillon) #7

Thanks @llenzi! I have submitted a bugfix for that problem on the q2-itsxpress code repo, hopefully that fix will be available some day!


(Adam Rivers) #8

Sorry for the delay on reviewing the pull request. The changes are available on pip now.

pip install --upgrade q2-itsxpress

#9

Thank you for your help!
@Adam_Rivers I have upgraded q2-itsxpress, but unfortunately my trimmedITS.qza still becomes 33 KB. It doesn’t matter whether I choose region ITS1, ITS2 or ALL. The files within the trimmed files seem empty. Subsequent dada2 gives the following error: An error was encountered while running DADA2 in R (return code 1), please inspect stdout and stderr to learn more.


(Luca) #10

Hi @Adam_Rivers,
I have no better luck with the update too. Tested on qiime2-2018.11 and qiime2-2019.1.
Installing collected packages: q2-itsxpress
Found existing installation: q2-itsxpress 1.7.2
Uninstalling q2-itsxpress-1.7.2:
Successfully uninstalled q2-itsxpress-1.7.2
Successfully installed q2-itsxpress-1.7.3

When I export the trimmed sequences I got:
“Exported paired-end-demux.trim.qza as SingleLanePerSamplePairedEndFastqDirFmt to directory TEST”

The resulting MANIFEST is still:
sample-id,filename,direction
mock-24,mock-24_0_L001_R1_001.fastq.gz,forward

(I’m testing with mock24 in mockrobiota database)
Thanks
Luca


(Adam Rivers) #11

ITSxpress currently only supports primer sets in the “normal” forward orientation where for ITS1, the forward primer is in the 18S and the reverse is in the 5.8S, or for ITS2 the forward primer is in the 5.8s and the reverse primer is in the 28S. Your mock data set uses the primers from Taylor et al. (2016) which are in the reverse orientation. This limitation is documented in the README section.

ITSxpress does not currently support reversed primer sets, but it is on the roadmap for the next incremental release, probably in May.


(Luca) #12

Sorry I did not realised that!
Thanks !
Luca


(Adam Rivers) #13

Sorry, It’s a weird limitation and presently there are no checks to let you know you are using incompatible primers.


(Luca) #14

Hi Adam, no problem and most of all thanks for your work, there is a need for your plug-in!
At least is was written and I should check this details more carefully!

Luca


#15

Thanks @Adam_Rivers, I also didn’t realise that q2-itsxpress used only forward reads.


(Nicholas Bokulich) #16

itsxpress can use both forward and reverse reads. The issue here is that it expects the forward reads to be in the same orientation as the reference sequences that it uses. Whether you are using single-end or paired-end sequences, orientation is important.


(Adam Rivers) #17

Thanks, I was just about to clarify that.