Merging ITS1 & ITS2 primers: a question about methods

[Antani (Edoardo Scali)]

Dear Qiime2 community,
I have a question about a data analyses procedure.

I have a set of 500 samples from whom ITS1 and ITS2 have been amplified.
I've analyzed them without merging these two dataset because I read that in this case, it could give false positive or create chimeric sequences.

I performed taxonomic classification on both ITS1 and ITS2 dataset with the Qiime release of UNITE database version 9 (2023-07-18) containing all Eukaryotic sequences.

After this step, I compared the ITS1 and ITS2 classification by looking at how many UNITE IDs were shared between the two dataset and I found that 350 ASVs have been classified as the same.

My question is: can I merge ITS1 and ITS2 if I remove these shared ASVs from one of the dataset?

I was thinking that maybe I will not be able to use those matrices that take phylogeny into account, but perhaps Jaccard or Bray-Curtis are still applicable.

Also, if this is a correct procedure, do you think that it mght be worthed useing just this database for the data analyses?

Thank you so much for your help and opinions!!

[colinbrislawn (Colin J Brislawn)]

@Antani

My question is: can I merge ITS1 and ITS2 if I remove these shared ASVs from one of the dataset?

Yes, but there's going to be big trade-offs involved in working with different regions.

Justing made a table about working with multiple regions and what to expect:

Using multiple 16s variable regions for analysis

Thanks for the tag @lizgehret! Yeah, I've spent a lot of time thinking about this in the last year. Attempting to write a paper, TBH, but its slower going than I expected. So, @Rakaya, like Liz said, the issue in directly combining the two regions is that you're not going to have an ID overlap if you only use the ASVs. Remember from ESVs should replace OTUs that the ASV ID is that single nucleotide sequence. ASVs that differ by a single nucleotide are going to be identified as different, and so…

@Antani

looking at how many UNITE IDs were shared between the two dataset and I found that 350 ASVs have been classified as the same.

This sounds like the column 'Genus-level taxa' from Justine's table.

[Antani (Edoardo Scali)]

Dear @colinbrislawn,
Thank you for your prompt response. I found Justine's table to be very informative and useful!