incomplete taxonomic assignment

Dear Community,

I am processing multi-amplicon sequencing data from Ion GS S5 sequencing platform. My basic approach involves DADA2 denoising followed by feature classification using classify-consensus-blast/vsearch.
After classification, I am getting partial taxonomic assignment ( till family level/ class level) eg- "d__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Enterobacterales;;;__" .
I understand this might be happening due to poor primer trimming/small amplicon length or some other reason associated with sequence quality. However, when I take the ASV sequence and try using blast (NCBI nr) I am able to pickup the genus level taxonomy correctly.

What I want to know is, if there is any other way to improve the taxonomic assignment, to get a genus level assignment (even with little low confidence) for most of my ASVs ??

I tried using vsearch separately for the such ASVs with 0.8 cutoff, and the results are not very convincing.

Thank you for your support!!!

Hi @Saptarathi_Deb,

I am assuming you are running with default settings against using either SILVA or Greengenes reference database?

A few things to note:

  1. You could be observing a limitation of the reference database being used (i.e. SILVA / Greengenes).
  2. Be wary of how BLAST hits are displayed on NCBI. That is, equivalent BLAST hits are arbitrarily sorted, and if you scroll down far enough you may find that there is an identical "hit" to a very different organism.
  3. Given 2, this is why we have classify-consensus-blast and classify-consensus-vsearch. Any hits which cannot be taxonomically resolved have their taxonomy truncated to the last common ancestor. This also applies to classify-sklearn too.

More information can be found here:

You can also try your hand at using RESCRIPt to make your own reference database for classifying your sequences:

This is the limitation of assigning taxonomy using short reads. However, you can use tools like q2-clawback to help improve things:

-Mike

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