Q2-itsxpress param help

Community Plugin Support
1

Hi Adam, thanks for developing that plugin. I have a doubt regarding the --p-taxa parameter. You have set it to "F" in the example, but I don't understand which I should use (ALL?). I´m processing ITS1 reads from clinical samples, sequenced by Miseq (2x300pb).

Thanks!

2

Generally the F parameter should be used unless you are targeting something specific like a mocrosporidian or something even more divergent like a marine diatom. Running ALL will not hurt anything but it will take much longer to process since more HMM models are searched. You can always run 1 sample both ways and compare the results.

2 Likes
3

OK, then I will run my sequences under the F mode. Thanks Adam!

4

Hello @Adam_Rivers !

I created a topic in the forum last week (Rarefying when having multiple runs - #16 by llenzi ) and they have recommended me to use your ITSxpress plugin.

I have had a glance at one of the first posts about the plugin (Q2-ITSxpress: A tutorial on a QIIME 2 plugin to trim ITS sequences) and I have a doubt before I use it.

The people that made the amplification of the samples decided to combine both ITS1 and ITS2 regions, so, how can I specified this in the --p-region parameter of the following command?

qiime itsxpress trim-pair-output-unmerged\
  --i-per-sample-sequences sequences.qza \
  --p-region ITS1 \
  --p-taxa F \
  --p-cluster-id 1.0 \
  --p-threads 2 \
  --o-trimmed trimmed_exact.qza

Thank you so much in advanced :slight_smile:

5

If you have paired-end reads the span the entire ITS1, 5.8s, and ITS 2 region you can use the --p-region ALL. But chances are you should not use this flag. Moat paired-end technologies would not span the entire region and either both ends would not be present or the reads would not merge. causing those reads to be thrown out,

What sequencing technology (e.e. Illumina miseq Paired End 2x300) and primer set are you using?

6

Hi @Adam_Rivers !

The sequencing technology we used was PGM from Ion Torrent, so it is no neither single nor paired-end... And the primers my colleagues used were:

ITS 1:
ITS1-30F (5´-GTCCCTGCCCTTTGTACACA-3´)
ITS1-217R (3´-TTTCGCTGCGTTCTTCATCG-5´)

ITS 2:
ITS-86F (5´-GTGAATCATCGAATCTTTGAA-3´)
ITS4R (3´-TCCTCCGCTTATTGATATGC-5´)

7

ITSxpress doesn't officially support ion torrent data and I've never tested ion torrent data but it could work.

I don't know what your amplicons look like but I'm guessing that there is only one read of the amplicon (single-ended) and that somehow the its1 and its2 amplicons have been concatenated together. Can you explain more about your library construction?

8

Hello @Adam_Rivers !

Sorry for the late response, but as I'm not aware of the process of the library construction, I had have to ask many things to the genomic platform manager.

The followed steps are the following:

  1. Two different amplifications were done for each sample, one for each ITS region (with the primers above).
  2. Those amplifications that show a band in the PCR step were send for sequenciation. Therefore, if the ITS1 amplification of sample1 showed a band in PCR but ITS2 did not, only ITS1 was sequenced for that specific sample. However, if both regions showed positive band, both amplified dna products were mixed in a tube and sequenced together. In conclusion, we will have some samples that only have reads from one region (either ITS1 or ITS2) and others that will have a mix of them.
  3. When it comes to the library construction, I attach a pdf file where the used kit is explained but, summing up, referring to what they have explained me...
    Besides the purification steps, a barcode and an adapter are attached to the reads of each sample, so each sample has its own identifier. Therefore, as I understand, for those samples where ITS1 and iTS2 have been combined, we will have reads from both regions with the same barcode identifier.

However, I don't know to what extent this affects, since the fastq files I use already have this barcode removed.

I know its a bit confusing..