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vmikk / NextITS

A pipeline for metabarcoding fungi and other eukaryotes with full-length ITS sequenced with PacBio

Home Page:https://next-its.github.io

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secondary analysis

splaisan opened this issue · comments

commented

Dear Vladimir,

I am finally starting to use your pipeline and fixed a few issues at my side that were crashing step2.
I now have al expected folders but do not find how to proceed and get classification (I now ran a manual blast) as well nice bar plots and also phylogeny plots.

I expect this can be done using Qiime2 but I am still not too familiar with it and which files need be imported to create the right objects for plotting.

Would you by any chance have a tutorial explaining the next steps for visualisation to obtain results similar to the pb-16S-nf pipeline does for 16S. Ideally, an extension to your current https://next-its.github.io/ doc would be perfect with results from some test data.

Thanks in advance

commented

Hello Stephane,

I'm sorry for the late reply.

Currently, NextITS does not perform taxonomy annotation of the resulting sequences. In our group, we primarily use BLASTn for this purpose (as it is the most sensitive classifier among all we have tested) in conjunction with the EUKARYOME database. However, BLAST is extremely slow and should be run in parallel on separate nodes if you have a large number of sequences to annotate. Additionally, the resulting table requires curation, which is not yet automated.

In general, since the output OTUs are in FASTA format, any taxonomy annotation tool should support it. We might consider adding a taxonomy annotation step, but we need to decide on the most viable approach.

Regarding the test data and documentation, updating them is certainly a good idea, and it is already on our to-do list.

With kind regards,
Vladimir

P.S. We have added support for asymmetric barcodes to NextITS.