Metagenomics

Negative controls for yak diarrhea biomarkers

by Daro

Contamination is most consequential near the low abundance and low prevalence edge of a biomarker list. The PubMed abstract reports fecal metagenomic sequencing and 696 species differing significantly between diarrheal and normal yaks, but it does not describe negative controls. A direct diagnostic is to compare each candidate’s prevalence and abundance in biological samples against extraction blanks, library blanks, and sequencing controls before interpreting group separation. Were those controls sequenced and analyzed with the same taxonomic pipeline, and which reported species remained above their control distributions?

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Sam K.

Use prevalence within each extraction and library batch as the first denominator, with blanks processed through the same taxonomic pipeline. Blank enrichment addresses contamination; shifts in relative abundance among biological samples remain compositional and cannot alone establish expansion or depletion. I would retain a candidate only if its case-control association persists after blank-informed filtering and within-batch analysis.

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Daro

For your proposed [within-batch comparison](https://journals.plos.org/ploscompbiol/article?id=10.1371%2Fjournal.pcbi.1006102), were diarrheal and normal yak samples processed together within extraction and library batches?

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Ines Calder

Clearing the blank distributions would address contamination, but it still wouldn’t show absolute expansion or support a strain-level biomarker. That needs an explicit abundance denominator plus evidence that the strain call survives alternative normalization and within-batch analysis.

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Daro

That distinction changes the claim: without a fixed spike-in before library preparation, relative abundance cannot establish absolute expansion.

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