Methods for normalizing microbiome data: an ecological perspective

McKnight, Donald T., Huerlimann, Roger, Bower, Deborah S., Schwarzkopf, Lin, Alford, Ross A., and Zenger, Kyall R. (2019) Methods for normalizing microbiome data: an ecological perspective. Methods in Ecology and Evolution, 10 (3). pp. 389-400.

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Abstract

1. Microbiome sequencing data often need to be normalized due to differences in read depths, and recommendations for microbiome analyses generally warn against using proportions or rarefying to normalize data and instead advocate alternatives, such as upper quartile, CSS, edgeR-TMM, or DESeq-VS. Those recommendations are, however, based on studies that focused on differential abundance testing and variance standardization, rather than community-level comparisons (i.e., beta diversity). Also, standardizing the within-sample variance across samples may suppress differences in species evenness, potentially distorting community-level patterns. Furthermore, the recommended methods use log transformations, which we expect to exaggerate the importance of differences among rare OTUs, while suppressing the importance of differences among common OTUs.

2. We tested these theoretical predictions via simulations and a real-world dataset.

3. Proportions and rarefying produced more accurate comparisons among communities and were the only methods that fully normalized read depths across samples. Additionally, upper quartile, CSS, edgeR-TMM, and DESeq-VS often masked differences among communities when common OTUs differed, and they produced false positives when rare OTUs differed.

4. Based on our simulations, normalizing via proportions may be superior to other commonly used methods for comparing ecological communities.

Item ID: 57742
Item Type: Article (Research - C1)
ISSN: 2041-210X
Keywords: Bray-Curtis, community comparisons, diversity, evenness, ordination, principal coordinates analysis, simulation
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Copyright Information: © 2018 The Authors. Methods in Ecology and Evolution. © 2018 British Ecological Society.
Additional Information:

A version of this publication was included as Chapter 5 of the following PhD thesis: McKnight, Donald T. (2019) Life finds a way: the recovery of frog populations from a chytridiomycosis outbreak. PhD thesis, James Cook University, which is available Open Access in ResearchOnline@JCU. Please see the Related URLs for access.

Research Data: https://doi.org/10.5061/dryad.tn8qs35, https://doi.org/10.18129/b9.bioc.metagenomeseq
Date Deposited: 27 Mar 2019 07:41
FoR Codes: 06 BIOLOGICAL SCIENCES > 0605 Microbiology > 060504 Microbial Ecology @ 100%
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