Protocol: PADOG: pathway analysis with down-weighting of overlapping sets against BugSigDB signatures

Tier: C (BugSigDB paper) · Type: atomic · Category: Statistical Analysis

What

Treat BugSigDB signatures as microbe sets and test them for enrichment using PADOG, which down-weights microbes appearing in many sets and uses sample permutation rather than set permutation. Input: a full expression/abundance matrix with sample labels, not a pre-thresholded list.

Why it matters

"Apply BugSigDB to my own data" is the most externally requested capability in the repository, and this is one of three genuinely different ways to do it.

Why this is its own protocol. The original draft bundled ORA, PADOG and CBEA into one issue with the method as a user choice. They have three different primary citations, three different null hypotheses, and three different input requirements — and PROTOCOL_STANDARD.md requires an atomic protocol to carry exactly one citation naming where the method was originally published. One protocol per method; a separate composite benchmarks them against each other, which is what Figure3.Rmd actually does.

Source material

• waldronlab/BugSigDBPaper — vignettes/Figure3.Rmd ("Obtain microbe signatures from BugSigDB", "Harmonize taxonomic level", "Enrichment analysis")
• Applied in: Geistlinger et al., Nat Biotechnol 2023, 10.1038/s41587-023-01872-y — cite this only as an application, never as the method's source

Scope

In: taxonomic-level harmonization of query and sets before anything else; minimum and maximum set size; multiple testing across sets; what a significant result licenses you to conclude. Method-specific: Why sample permutation preserves the correlation structure between microbes that set permutation destroys; the down-weighting formula; the minimum number of samples for permutation to be meaningful.

Out: producing the differential-abundance input; comparing against the other two methods (that is the benchmark composite).

Frontmatter starting point

type: "atomic"
category: "Statistical Analysis"
citation: ""   # Tarca et al. 2012, BMC Bioinformatics (10.1186/1471-2105-13-136) — VERIFY
tags: [enrichment, microbe-sets, padog, bugsigdb]

Acceptance criteria

• Cites the paper that proposed the method, not the BugSigDB paper
• Rank harmonization is a required step with defined behavior for unmatched taxa
• The universe/background and set-size bounds are explicit
• States what the test's null hypothesis actually is, in words

Cite the method's origin, not its users

PROTOCOL_STANDARD.md is explicit: an atomic protocol carries "strictly 1 citation... corresponding
to the primary literature where the method was originally published." Find the paper that proposed
the method. Do not cite a paper that merely applied it — including the BugSigDB and curatedMetagenomicData
papers, which are the source of the analysis these protocols were extracted from but almost never the
source of the method.

Tracing a method back to its first publication is real work, and it is part of the task, not a
formality. Three things to expect:

• Candidate DOIs in this issue are leads, not answers. Anything marked VERIFY has not been checked.
• Some methods predate modern citation practice or have no single identifiable origin. If that is
  genuinely the case, say so in the pull request rather than reaching for a convenient recent paper.
  Raise it as an issue in waldronlab/agent-protocol-standard — the standard may need a way to express
  "classical method, no primary source".
• If you cannot name one paper that proposed everything the protocol does, it is more than one
  protocol. That test has now split four protocols out of this batch: enrichment into three methods,
  filtering from transformation, LODO from random forest, and PERMANOVA from ANOSIM.

Where the lab's own paper genuinely did propose the method — the oral-to-gut score, and LODO
cross-validation in Pasolli et al. 2016 — citing it is correct. That is the exception, not the pattern.

Before you start

Read CONTRIBUTING.md and
PROTOCOL_STANDARD.md.
The format is defined in the standard repo, not this one. Protocols are prose, not code: they say what to do
and why, precisely enough that two people — or two agents, in two languages — get the same answer. The existing
independent-filtering-variance
protocol is the model to imitate for tone and level of detail.

Validate locally before opening the PR:

git clone https://github.com/waldronlab/agent-protocol-standard.git
Rscript agent-protocol-standard/scripts/validate-protocol.R protocols

Read CONTRIBUTING.md and PROTOCOL_STANDARD.md first, then use protocols/independent-filtering-variance/protocol.md as the style model. Check Figure3.Rmd for the cited analysis and verify the primary PADOG paper rather than relying on the candidate DOI. Done means a protocol with one method-origin citation, explicit harmonization, universe and set-size rules, and the null hypothesis and acceptance criteria stated clearly; validate with the provided Rscript command.