ITSxpress outputs only forward read file

I am trying to analyze my fungal paired-end ITS2 amplicon (ITS86F/ITS4 primers) data and would like to use the ITSxpress tool.
I run it on the demultiplexed imported .qza file
qiime itsxpress trim-pair --i-per-sample-sequences demux-paired-end_test_low_reads.qza --p-region ITS2 --o-trimmed itsxpresslow.qza --p-threads 15
When I export the output file (qiime tools export) it only has the R1 reads fastq.gz files (the initial demux file has both R1 and R2) and when trying to use dada2 denoise on the output file it gives an error : TypeError: Argument to parameter ‘demultiplexed_seqs’ is not a subtype of SampleData[PairedEndSequencesWithQuality].

Why do my R2 files get lost?

Thank you in advance!

HI @rahel_park,
It looks like q2-itsxpress trim-pair outputs a SampleData[SequencesWithQuality] artifact (and you can always determine the type of an artifact by using qiime tools peek, by the way). So I believe the output reads are already merged and would be denoised as single-end reads, e.g., with dada2 denoise-single.

@Adam_Rivers can give us more information on the typical workflow!

I hope that helps!

Thanks for the reply,
Indeed when I do the dada2 denoise-single and check later the rep-seqs then it looks like the files were merged even though the fastq files in the .qza file are named only *_R1_001.fastq.gz
I guess this solves my problem :slight_smile:

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Hi @rahel_park,

That’s correct, ITSxpress only outputs merged sequences. Sequences that do not merge are removed. If you want to do you own merging you can do that beforehand and pass ITSxpress single-ended sequences. After ITSxpress you need to use dada2 denoise-single I’ll make this clearer when I write the documentation for the QIIME2 site this week.

Hi @Adam_Rivers ,
Thanks for the explanation.
I did as well parallel the analyses 1) itsxpress and then taxonomy with unite database qiime_ver7_dynamic_s_01.12.2017 2) no itsxpress, just cutadapt and then taxonomy with unite database qiime_ver7_dynamic_s_01.12.2017_dev (as I understood that in the developer version the UNITE has still the flanking ribosomal genes there…)
When using itsxpress I have a lot more reads after denoising and as well final sequence variants (~double compared to without itsxpress).
What could be the reason for such a big difference? It’s a bit complicated to wrap my head around it…

This is interesting, I’d like to look into it more. Would you be willing to share your commands and qiime files with me? If so, PM me with details about how to download the data.


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