Protocol: derive a reference species set for a body site or sample group by prevalence

Tier: D (cMD paper) · Type: atomic · Category: Curation & Data Preparation

What

Given any labelled group of samples — a body site, a population, a disease cohort, an environment — select the species prevalent enough within that group to serve as its reference set, and publish the result as a versioned, citable list.

Why it matters

Generalized from the oral-specific draft, per review. The original version derived the oral species list and nothing else, which made it useful exactly once. The procedure is not oral-specific: pick a reference sample group, apply a prevalence rule, publish the list. Stated generally it supports a skin set, a vaginal set, a healthy-adult-gut set, a population-specific set — and the 305-species oral list becomes one worked example rather than the whole point.

It is also the generator for a family of "enrichment score" analyses. Once a reference set for group X exists, the scoring protocol can measure X-typical organisms in samples from elsewhere. Oral-to-gut is the instance the paper published; there is no reason it should be the only one.

Source material

• waldronlab/curatedMetagenomicDataAnalyses — cMD3_paper_analyses/oral_enrichment/01-estimate_oral_enrichment.py, python_tools/oral_introgression_score.py (evaluate_oral_species)
• Worked example: 10.1038/s41467-025-66888-1 — 305 species prevalent in ≥ 1% of oral samples

Scope

In: how the reference sample group is defined and what makes it adequate (size, diversity of source studies, whether a single cohort can ever be sufficient); the prevalence rule and threshold, and its relationship to the detection floor of the profiler used; the profiler and reference-database dependency — a list derived under one taxonomic backbone is not directly usable under another, and the protocol must say how that is recorded; the versioning and publication scheme, so that scoring protocols can pin a specific list; what must be re-validated when a list is regenerated.

Out: computing scores from the list (that is oral-to-gut-enrichment-score and its future siblings).

Frontmatter starting point

type: "atomic"
category: "Curation & Data Preparation"
citation: "10.12688/f1000research.8986.1"   # prevalence as the selection criterion; but see note
protocols_used:
  - name: "prevalence-filtering"
tags: [reference-set, prevalence, body-site, species-list, reference-data]

Citation needs thought. The prevalence criterion itself traces to Callahan et al. 2016, but using prevalence within a reference group to define a marker set for a different group may be a distinct contribution — plausibly the cMD paper's own. Establish which and say so.

Acceptance criteria

• Stated for an arbitrary sample group; oral appears only as a worked example
• Rerunning it on the paper's oral reference set reproduces the 305-species list
• The profiler / reference-database dependency is recorded in the published list
• Defines the versioning scheme that scoring protocols pin against

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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, PROTOCOL_STANDARD.md, and protocols/independent-filtering-variance/protocol.md first. Review the cited oral-enrichment scripts and trace the primary literature for the prevalence method, then write the general protocol with the profiler/database, versioning, and regeneration requirements. Validate it with Rscript agent-protocol-standard/scripts/validate-protocol.R protocols and confirm the oral example reproduces the 305-species list.