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What did the term OTU refer to before the era of DNA sequencing?

OTU -> operational taxonomic unit is an operational definition Introduced in 1963 by Robert R. Sokal and Peter H.A. Sneath to define simply the group of organisms currently being studied. An OTU is a pragmatic definition to group individuals by similarity, equivalent to but not necessarily in line with classical Linnaean taxonomy or modern evolutionary taxonomy.

The definition is intentionally really vague: the object of the observation could be an organism or a taxonomic group. It is up to scientists to define what an OTU is.

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What is an OTU in DNA metabarcoding era?

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DNA metabarcoding data analysis

Every time we analyse NGS data we need to face the issue related to sequencing errors.

For instance in genomics and transcriptomics we usually trim-out low quality region of reads.

In DNA-metabarcoding we need to account for:

Amplification errors:

Substitution
Chimera

Sequencing errors

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DNA metabarcoding data analysis

OTU picking is the bioinformatic procedure applied for several years to treat DNA-metabarcoding data, with two general purposes:

reduce the effect of amplification and sequencing noise
reduce computational requirements.

Historically, OTUs represent groups of sequences with a similarity over a defined threshold, usually 97%.

We will briefly discuss how this threshold was defined and the reason why we need to overtake the idea of a fixed threshold.

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What is a species?

A species is often defined as the largest group of organisms in which any two individuals of the appropriate gender or mating type can produce a fertile offspring, typically by sexual reproduction.

Ciccarelli FD et al (2006) Toward automatic reconstruction of a highly resolved tree of life. Science. 3;311(5765):1283-7.

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Some definitions

For many decades, bacterial species definition was based on genome similarity measured by using DNA-DNA hybridization (DDH). Two genomes are conspecific (belongs to the same species) if their DDH ≥ 70% (Wayne et al, 1987).
Today, the genome similarity is measured as ANI (average nucleotide identity). Two genomes are conspecific if their ANI ≥ 95%.
Actually, microbiologist use a combined approach to define bacterial species based on genetic and phenotypic tracts (polyphasic taxonomy)

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OTU

With the advent of sequencing techniques and the availability of full-length 16S rRNA sequences, the OTU was defined using a 97% identity threshold.
This value has been shown to correspond to a DDH value ≥ 70% (Stackebrandt and Goebel, 1994) which has been previously accepted as working species for bacteria.

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OTU

Relationship between the rRNA 16% identity percentage and genomic DNA hybridization between different couples of microorganisms belonging to the Bacteria domain.

Orange area: 16S identity % and % of genomic similarity, higher than 97% and 70% respectively. Same species.

Blue area: 16S identity % higher than 97% but genomic similarity lower than 70%. Species sharing a similar 16S but not the same genetic content.

Green area: 16S identity % and % of genomic similarity, lower than 97% and 70% respectively.

TAKE HOME MESSAGE: The 16S gene may be very similar but does not always reflect the entire genome.

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TAKE HOME MESSAGE: The 16S rRNA gene may be very similar but does not always reflect the entire genome.

Thus be careful using predictive software such as:

-1) PICRUSt2 for prediction of metagenome functions

-2) Tax4Fun2: prediction of habitat-specific functional profiles and functional redundancy based on 16S rRNA gene sequences

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