# Rarefaction in mothursop

**URL:** <https://forum.mothur.org/t/rarefaction-in-mothursop/22372>\
**Category:** Theory behind mothur\
**Created:** [October 13, 2025, 5:37pm UTC](https://forum.mothur.org/t/rarefaction-in-mothursop/22372 "2025-10-13T17:37:43Z")\
**Posts on this page:** 3\
**Page:** 1

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**Author:** ![mainly.microbe](https://avatars.discourse-cdn.com/v4/letter/m/77aa72/32.png) [@mainly.microbe](https://forum.mothur.org/u/mainly.microbe)\
**Post date:** [October 13, 2025, 5:37pm UTC](https://forum.mothur.org/t/rarefaction-in-mothursop/22372/1 "2025-10-13T17:37:43Z")

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Hello! I previously asked when to use the sub sampled data and sadly I am still confused. I was told “Things like amova/homova/pcoa/shannon/simpson/richness are calculated using dist.shared and summary.single using rarefaction.” and I understood that as we DO use the sub sampled data for these things.

In the mothur SOP The rarefaction step does not use the sub sampled data but to do the other alpha diversity indices in the next step, the sub sampled data IS used. I am just confused when to use the sub sampled file still.

rarefaction.single(shared=final.opti\_mcc.shared, calc=sobs, freq=100) WHY NOT HERE?

summary.single(shared=final.opti\_mcc.shared, calc=nseqs-coverage-sobs-invsimpson, subsample=T) HERE

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**Author:** ![pschloss](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/pschloss/32/4_2.png) [@pschloss](https://forum.mothur.org/u/pschloss)\
**Post date:** [October 14, 2025, 12:45pm UTC](https://forum.mothur.org/t/rarefaction-in-mothursop/22372/2 "2025-10-14T12:45:52Z")

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Hi there

Sorry for the confusion. subsampling is a single step of rarefaction. So, it wouldn’t make sense to use it in `rarefaction.single()` since you’d get the same result every step since the data would have already been subsampled.

I would only suggest using the subsampled shared file with things like lefse, metastats, get.communitytype.

Pat

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**Author:** ![system](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/system/32/2_2.png) [@system](https://forum.mothur.org/u/system)\
**Post date:** [October 24, 2025, 12:46pm UTC](https://forum.mothur.org/t/rarefaction-in-mothursop/22372/3 "2025-10-24T12:46:20Z")

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