Rarefaction

Rarefaction (even sampling depth) is used to normalize for unequal sequencing depth across samples before calculating diversity metrics. The alpha rarefaction curves help you identify a depth where diversity estimates have stabilized, allowing you to retain as many samples as possible while excluding only those with very low read counts.

Alpha Rarefaction Curves

This command calculates multiple alpha diversity metrics (Faith's PD, observed features, Shannon entropy) at rarefaction depths ranging from 1 to --p-max-depth and plots them as curves:

qiime diversity alpha-rarefaction \

--i-table table_nomitochloro.qza \

--m-metadata-file metadata_q2_workshop.txt \

--p-max-depth \

--o-visualization alpha_rarefaction_curves.qzv

Hint Params

Check →

Open table_nomitochloro.qzv from the previous step. In the interactive table summary, find the value around the 95th percentile of per-sample read counts. Setting max depth too low truncates the curves before they plateau; too high wastes computation. For this dataset, that value is 5500.

--p-max-depth, Maximum rarefaction depth (integer). The command calculates diversity at multiple depths from 1 to this value and plots them as curves. Should be approximately the 95th percentile of per-sample read counts from your table summary.

Choosing a Max Depth

Set --p-max-depth to approximately the 95^th percentile of your per-sample read counts, which you can identify in the table_nomitochloro.qzv summary from the previous step. Setting it too high wastes computation; too low and your curves may not plateau.

Interpreting the Curves

Open alpha_rarefaction_curves.qzv in QIIME2 View and look for the depth where diversity metrics plateau, this is where additional reads no longer add new diversity information.

Choosing a Rarefaction Depth Reset

Identified the plateau depth for all three metricsFaith's PD, observed features, and Shannon entropy should all stabilize Checked how many samples fall below the chosen depthSamples below this threshold will be excluded from diversity analyses Balanced depth vs. sample retentionDeeper depth retains more reads per sample but drops more samples Confirmed rarefaction depth of 1,500 readsUsed in the core metrics step for this workshop dataset

Workshop Sampling Depth

For this dataset we use a sampling depth of 1,500 reads in the core metrics step. This retains the majority of samples while excluding those with insufficient sequencing coverage.

Outputs

File	Type	Description
alpha_rarefaction_curves.qzv	Visualization	Alpha diversity vs. rarefaction depth

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