Hello,

OTU means Operational Taxonomic Unit, and it represents, as suggested by the name, an operational definition of a taxon, in contrast with a more classical taxonomic definition, normally based on morphology or physiology. As you might imagine, it would be very difficult to classify millions of microorganisms with the classical approach, thus, when you work with high-throughput sequence-based analyses you need to delineate taxonomy that do not depend on the visual and singular characterization of microorganisms. A typical OTU for bacteria is defined as those organisms with a sequence similarity percentage of 97% or higher of a phylogenetic gene marker, such as the ssu_rRNA or 16S gene. In summary, you might interpret each OTU as a bacterial species, although having in mind that there are exceptions to this rule of course (i.e. within an OTU you can have multiple bacterial species).

In the plot you show, you have samples, named C1, C2, C3 and so on. Maybe they represent different microbiota from different patients in different conditions. The plot is telling you that the sample C3 is the more diverse, as the number of different species (OTUs) increase more than in other samples, with increasing number of sequences. In contrast the sample C9 is the less diverse, as the number of species does not increase very much regardless of the number of sequences that you analyze.

I hope that helps.

Best regards.

Hai, I feel this link should help you in a simplified manner to interpret rarefaction curves http://www.biosym.uzh.ch/modules/models/Biodiversity/MeasuresOfBioDiversity.html

Best wishes

Hello,

OTU means Operational Taxonomic Unit, and it represents, as suggested by the name, an operational definition of a taxon, in contrast with a more classical taxonomic definition, normally based on morphology or physiology. As you might imagine, it would be very difficult to classify millions of microorganisms with the classical approach, thus, when you work with high-throughput sequence-based analyses you need to delineate taxonomy that do not depend on the visual and singular characterization of microorganisms. A typical OTU for bacteria is defined as those organisms with a sequence similarity percentage of 97% or higher of a phylogenetic gene marker, such as the ssu_rRNA or 16S gene. In summary, you might interpret each OTU as a bacterial species, although having in mind that there are exceptions to this rule of course (i.e. within an OTU you can have multiple bacterial species).

In the plot you show, you have samples, named C1, C2, C3 and so on. Maybe they represent different microbiota from different patients in different conditions. The plot is telling you that the sample C3 is the more diverse, as the number of different species (OTUs) increase more than in other samples, with increasing number of sequences. In contrast the sample C9 is the less diverse, as the number of species does not increase very much regardless of the number of sequences that you analyze.

I hope that helps.

Best regards.

Salvador Ramirez-Flandes

Is it possible to share some straightforward references (paper or book) regarding rarefaction and normalization?

Thanks

Mehrdad Rabiei, I can recommend you this article:

McMurdie, P. J., & Holmes, S. (2014). Waste not, want not: why rarefying microbiome data is inadmissible. PLoS computational biology, 10(4).

Article Waste Not, Want Not: Why Rarefying Microbiome Data Is Inadmissible

in which the authors argues that rarefaction is the worst option for normalizing microbiome data. Best regards.

Salvador Ramirez-Flandes Deviram Garlapati Thank you so much!!

Hi, the rarefaction plot is showing the number of indivudal OTUs identified in a given rarefaction. It will divide your sample in many samples with varying number of reads (Rarefied samples) and use it to calculate an alpha-diversity metric accross this range of sample sizes. The idea is to identify the minimum sample size where the number of OTUs reach a plateau, which means that it doesn't matter if you increase the number of reads, the OTU number will not increase anymore.

Hmmm. let me process this for awhile!