6. Alpha rarefaction plotting - DianaCarolinaVergara/16S-rRNA-Analysis GitHub Wiki

Alpha-rarefaction Plotting

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.

Alpha rarefaction plot demonstrates phylotype diversity in the samples. Shown are the selected metric you want.

Numbers of different Operational Taxonomic Units (OTUs), Shannon, etc.

as a function of the numbers of sequences analyzed and generated with QIIME.

Some examples of alpha-rarefaction plots:

1. Filtered by name

2. Or filtered by BarcodeSequence

Code:

qiime diversity alpha-rarefaction \
  --i-table table-dada2.qza \
  --i-phylogeny insertion-tree.qza \
  --p-max-depth 65000 \
  --m-metadata-file sample_metadata.txt \
  --o-visualization alpha-rarefaction.qzv

And with this graphic you select the Sequencing Depth (where the asymptote is reached on the graph).

qiime diversity core-metrics-phylogenetic \
  --i-phylogeny insertion-tree.qza \
  --i-table table-dada2.qza \
  --m-metadata-file sample_metadata.txt \
  --p-sampling-depth 15000 \
  --output-dir core-metrics-results