Question: Normalization for microbiome 16s sequence analysis.
0
gravatar for ariel
28 days ago by
ariel40
ariel40 wrote:

The way I understand things, normalization (such as in DeSeq2, EdgeR, etc.) serves two purposes: 1) Model the "real" abundance in the original samples from the read counts, 2) Make the abundance distributions conform to the needs of statistical analysis by removing heteroskedasticity, dependence, dispersion, etc.

It has been stated many times here that it is very difficult to reproduce the fold-change you get from DeSeq2 by extracting the normalized counts, but you can come close. Taken at face semantic value, "fold change" sounds like it should refer to the ratio of the "real" abundance; the fold change of "actual" expression or "actual" community representation.

So if the normalized (or normalized + VST, or normalized + MLE) abundance better represents the "real" abundance, then shouldn't I use the normalized counts for ALL of my analysis steps:

  • Alpha diversity
  • Beta diversity
  • F2B ratio
  • IgA sorting analysis
  • Other regression analysis
  • etc.
rna-seq miseq 16s microbiome • 151 views
ADD COMMENTlink modified 18 days ago • written 28 days ago by ariel40

Hi ariel, did you get any further?

if the normalized (or normalized + VST, or normalized + MLE) abundance better represents the "real" abundance, then shouldn't I use the normalized counts for ALL of my analysis steps?

My take is "Mostly yes, but it depends". I do use the normalized counts for pretty much everything, especially when comparing samples. When you look at each sample individually, there's no harm to use the original count data. Important is to know about the limitations and compare results when in doubt.

ADD REPLYlink written 20 days ago by Carambakaracho1.4k
2
gravatar for ariel
18 days ago by
ariel40
ariel40 wrote:

@Carambakaracho -- thanks for checking back! In fact, I received no response here or on http://bioinformatics.stackexchange.com/. This surprised me because it seems like a very important question.

Fortunately, I did get some valuable response at the QIIME2 forum that you might find interesting.

https://forum.qiime2.org/t/normalization-for-microbiome-16s-sequence-analysis/10398/8

ADD COMMENTlink written 18 days ago by ariel40

Thanks for sharing. That's a pretty interesting read, until today, I was an advocate of Susan Holmes' Waste not Want not concept

ADD REPLYlink written 17 days ago by Carambakaracho1.4k
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