Identification of Acanthamoeba in 18S rDNA analysis

General Discussion
1

Hello!

I am conducting an analysis of 18S rDNA from a set of samples of amoebas isolated from water.

In this sample, the presence of Acanthamoeba species is known, as a qPCR for Acanthamoeba has been performed previously, and a large portion of the samples tested positive.

However, when performing taxonomic analysis using Qiime2 and the Silva database, Acanthamoeba was not identified in any of the samples.

Right now, we are testing the PR2 database, as it is more focused on eukaryotic classification and contains more Acanthamoeba sequences as well as other eukaryotes.

Nevertheless, we have tested this database (PR2) with other samples that were positive for Acanthamoeba, but in the latest version of PR2, Acanthamoeba was still not identified.

Is this a widespread issue when identifying Acanthamoeba with these databases? Or is it more likely due to some sequencing-related issue, such as the primers used?

Do you have any recommendations regarding databases or other aspects to improve Acanthamoeba identification using this technique?

Best regards, and thanks in advance,

Carla

2

Hi @Carla,

I am not too familiar with issues identifying Acanthamoeba species. But I'm generally not too surprised as rRNA genes are actually quite conserved, even the "variable" regions can be quite conserved among some taxa. Most of the time we're lucky to get genus level identification with short read SSU sequences.

What primer pair was used? Do you know if these primer pairs are ideally preferential to target the Acanthamoeba? or are were "universal primers" used?

Also, have you tried extracting the variable region of interest from the reference database? You can use qiime feature-classifier extract-reads ... to do this. How many of them have different taxonomic labels but identical / nearly identical sequences? This is quite common, and can partially explain why the taxonomic resolution can be poor... If multiple and equally viable "hits" to different nearly identical reference sequences are encountered (with differing taxonomy)... often the lowest common ancestor taxonomy is returned.

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