I am attempting to analyze a dataset generated from fungal ITS sequences. I have ITS1 (from ITS1F and ITS2R primers) and ITS2 (from ITS3F and ITS4R) for each sample.
What should i do ?
Merging this region by merging fastq files ? but, how find which region formed an OTU ?
Or realize the analysis separately ?
Thanks a lot,
Definitely analyze separately. These regions do not actually overlap, as far as I know, and even if there is a small region of overlap it would be in the conserved 5.8S region so you would effectively just create chimeras.
Results will differ between ITS1 and ITS2, because each primer/region is subject to its own biases, so I would also discourage you from merging data even after assigning taxonomy to these sequences. Alpha diversity, even beta diversity results will probably differ a bit between regions.
It may be worth comparing the results, e.g., to determine whether one has deeper taxonomic assignments or higher alpha diversity and hence might be more useful to you. However, unless if you are sequencing mock communities or other samples with known composition, you will not really know which region is actually giving better results.
Just analyze ITS1 and ITS2 separately and do not try merging at any point…
I hope that helps!
You are very clear, thank you very mush
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