incomplete taxonomic assignment

[SoilRotifer (Mike Robeson)]

Hi @Saptarathi_Deb,

@Saptarathi_Deb

My basic approach involves DADA2 denoising followed by feature classification using classify-consensus-blast/vsearch.

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

@Saptarathi_Deb

However, when I take the ASV sequence and try using blast (NCBI nr) I am able to pickup the genus level taxonomy correctly.

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:

Consensus blast taxonomy strings

Hi John, What you are observing is a peculiarity of the greengenes database, not of classify_consensus_blast. Your interpretation of the first sequence is correct (more notes on “confidence” below). However, the second sequence is actually being classified to species level by classify_consensus_blast — however, it is indicating that the top matches are all to greengenes reference sequences that are annotated in greengenes with empty family, genus, and species annotations. Greengenes contains a …

BLAST+ taxonomy classifier

Hi @microbiomeAnalyst, Good questions! Sure, if those are the sequences you want to taxonomically classify. No, this is ANY reference database in the appropriate format (see the SILVA and greengenes reference files linked to from here). Note that BLAST is the name of the algorithm used for alignment, and is not the same thing as NCBI-BLAST (a webtool implementation of BLAST). The outputs of this classifier (which performs LCA classification on top of BLAST alignment) is also not the same th…

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

Using RESCRIPt to compile sequence databases and taxonomy classifiers from NCBI Genbank

This tutorial will describe how to create custom reference databases from NCBI Genbank using RESCRIPt. Read more about RESCRIPt here: https://forum.qiime2.org/t/processing-filtering-and-evaluating-reference-sequence-data-with-rescript/15494/7 If using NCBI Genbank data, please be aware of the NCBI disclaimer and copyright notice. Citation: If you use RESCRIPt or any RESCRIPt-processed data in your research, please cite the following pre-print: Michael S Robeson II, Devon R O'Rourke, Benjami…

@Saptarathi_Deb

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 ??

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

Using q2-clawback to assemble taxonomic weights

Note: This guide assumes you have installed QIIME 2 using one of the procedures in the install documents. This tutorial extends the q2-feature-classifier tutorial to show how you can improve taxonomic classification accuracy by assembling ecologically informed taxonomic weights for your samples using q2-clawback. This approach is described in this article: https://doi.org/10.1038/s41467-019-12669-6 We will retrain the naive Bayes classifier from the q2-feature-classifier to classify the ski…

-Mike