Species classification error


I am doing microbiome analysis using qiime2’s “Moving Pictures” tutorial.

I found a problem during the analysis process.

When analyzing the two samples A and B, when looking at the taxonomy results, the results were classified up to several species.

A wide variety of species came out, and among them we were able to confirm the species we were looking for.

However, when analyzing multiple samples by adding several other samples to the data of two samples A and B,

I used the same option value, DB

Almost only the kingdom level, or mitochondria, etc.

Only two samples were analyzed and no meaningful species could be found.

I am wondering what I did wrong and if there is any way to improve it



Later, when we analyzed several samples, we found a variety of species.

There, only the sample name was modified and the same option and the same command were executed.

Only the results that seemed meaningless were obtained, such as those classified up to the kingdom level or classified as mitochondria.

I don’t know if the results are different depending on the server situation

Welcome to the forum, @hwkim!

None of the classifiers in q2-feature-classifier generate “random” results or give different answers each time you run them, unless if you change the reference sequences… or if there is something wrong with the query sequences, e.g., they are in mixed orientations. Since you say everything else was kept the same (e.g., reference), I suspect your reads must be mixed orientations… the classify-sklearn method assumes that all reads are in the same orientation.

You can use RESCRIPt to orient your reads prior to classification:

Alternatively, you can try the VSEARCH-based classifier, which is not sensitive to read orientation. That would be worth doing as a quick test before installing RESCRIPt and reorienting your sequences.

Let us know what you find!


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