Alpha diversity
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11-Aug-2018 New paper describes octave plots for visualizing alpha diversity.

12-Jun-2018 New paper shows that one in five taxonomy annotations in SILVA and Greengenes are wrong.

18-Apr-2018 New paper shows that taxonomy prediction accuracy is <50% for V4 sequences.

05-Oct-2017 PeerJ paper shows low accuracy of closed- and open-ref. QIIME OTUs.

22-Sep-2017 New paper shows 97% threshold is wrong, OTUs should be 99% full-length 16S, 100% for V4.

24-Nov-2016
UPARSE tutorial video posted on YouTube. Make OTUs from MiSeq reads.

 

USEARCH v11

Alpha diversity


See also
  Alpha diversity metrics
  Octave plots
  Interpreting diversity metrics
  Recommended alpha and beta metrics
  Comparing alpha diversity between groups
  Statistical significance of diversity differences
  Rarefaction
  alpha_div command
  alpha_div_rare command

Alpha diversity is the diversity in a single ecosystem or sample. The simplest measure is richness, the number of species (or OTUs) observed in the sample. Other metrics consider the abundances (frequencies) of the OTUs, for example to give lower weight to lower-abundance OTUs. See alpha diversity metrics. The abundance distribution can be visualized using an octave plot

Interpretation
It is important to keep in mind that NGS amplicon sequencing cannot reliably measure frequencies or presence / absence of OTUs, so the biological meaning of alpha diversity metrics developed for traditional ecology is unclear / misleading / difficult to interpret.

Estimators
Some rare species may not have been observed. An alpha diversity estimator attempts to extrapolate from the available observations (reads) to the total number of species in the community. The best-known estimator for NGS OTUs is Chao1. In my opinion, estimators cannot be usefully applied to NGS OTUs because rare species are underrepresented if an abundance threshold is used (e.g., discarding singletons), and regardless the number of spurious OTUs increases at low abundances. The low-abundance tail of the distribution is therefore highly uncertain, and attempting to extrapolate makes no sense.

Rarefaction
The goal of rarefaction is to get an indication of whether enough observations have been made to get a good measurement of an alpha diversity metric. This is done by making a rarefaction curve which shows the change in a metric as the number of observations increases. If the curve converges to a horizontal asymptote, this indicates that further observations (i.e., more reads) will have little or no effect on the metric. As with estimators, the asymptote of a rarefaction curve depends on the low-abundance tail of the distribution, and is therefore of dubious value when applied to NGS reads. The number of OTUs is almost certain to increase with more reads due to errors, even if all species in a sample have been accounted for, and it is therefore almost certain that the rarefaction curve will converge to a positive slope.

Units of measurement
Confusingly, alpha diversity metrics often use different units. Sometimes the meaning is not obvious (entropy!?), and metrics with different units cannot be compared with each other. For example, the popular Shannon index is a measure of entropy where the unit is bits of information if the logarithms are base 2, but people sometimes use natural logarithms (base e) or base 10. None of these variants of the Shannon index have an obvious connection to the number of OTUs, and people often do not say which variant they used, so the numerical values are difficult to interpret.

Effective number of OTUs
Metrics using unfamiliar units can be interpreted and comparied by converting to an effective number of species.