Omicverse Microbiome Meta Analysis

OmicVerse Microbiome — Cross-cohort Meta-analysis

Goal

Take per-study microbiome AnnData objects (one per cohort, each from the 16S amplicon skill), stitch them into a single cross-cohort AnnData with combine_studies, then run per-study DA → cross-study meta-analysis with meta_da. Output is a single feature-level table with combined effect size, combined SE, p-value, BH-FDR, per-study count, and Cochran's I² heterogeneity — distinguishing features whose effect replicates across cohorts from cohort-specific signals.

The skill assumes the meta question is "does this contrast hold across cohorts?", not "is there an effect in any cohort". The latter is just running per-study DA and unioning hits — call that out as the wrong tool.

Quick Workflow

1. Load (or build) per-cohort AnnData — same conventions as the 16S amplicon skill (samples × ASVs, 7-rank SINTAX in var, obs[group_key] for the contrast).
2. Combine: ov.micro.combine_studies([a, b, c], study_names=[...], rank='genus', study_key='study') → AnnData — collapses ASVs to a common rank (genus by default), unions features across studies (zero-fills cohort-specific features), tags obs['study'].
3. Diagnostic Beta + PCoA on the combined object: confirms whether study effect dominates the disease effect — a useful sanity check before running meta_da. If PC1 colour-maps cleanly by study, the cohort effect is large and meta_da's random-effects pooling is doing real work.
4. Per-study top hits (optional, for sanity): run DA(a_g).deseq2(...) on each study independently, take top-5 by p-value; check whether the planted / known-true features appear in every cohort.
5. Meta-analysis: ov.micro.meta_da([a, b, c], study_names=[...], group_key, group_a, group_b, method='deseq2'|'wilcoxon'|'ancombc', rank='genus', min_prevalence=0.1, combine='random_effects'|'fixed_effects') → pd.DataFrame with feature, combined_lfc, combined_se, z, p_value, fdr_bh, n_studies, I2.
6. Plot the result: forest-style barh of top features (combined LFC ± 1.96·SE), colour-coding planted vs. cohort-specific vs. shared-noise features.
7. Heterogeneity scatter: combined LFC × I² — high-I² features (>0.75) are reproducibly different in direction between cohorts (suspicious); low-I² features (<0.25) are coherent. Use this to flag which "significant" hits actually replicate.
8. (Optional) Method × meta benchmark: re-run meta_da with method cycled over 'wilcoxon' / 'deseq2' / 'ancombc', compare hit counts and the rank of known-true features across methods.

Interface Summary

ov.micro.combine_studies(
    studies: Sequence[AnnData],
    study_names: Optional[Sequence[str]] = None,
    rank: Optional[str] = 'genus',         # collapse to this rank first; None keeps ASVs
    study_key: str = 'study',              # column name in combined obs
    min_prevalence: float = 0.0,           # additional filter applied after concatenation
) -> AnnData

ov.micro.meta_da(
    studies: Sequence[AnnData],
    group_key: str,
    group_a: Optional[str] = None,
    group_b: Optional[str] = None,
    method: str = 'deseq2',                # 'wilcoxon' | 'deseq2' | 'ancombc'
    rank: Optional[str] = 'genus',
    min_prevalence: float = 0.1,
    combine: str = 'random_effects',       # or 'fixed_effects'
    study_names: Optional[Sequence[str]] = None,
    **method_kwargs,
) -> pd.DataFrame
# columns: feature, combined_lfc, combined_se, z, p_value, fdr_bh,
#          n_studies, I2, plus per-study lfc / se / pvalue columns

Boundary

Inside scope:

• Combining 2+ per-study AnnDatas at any taxonomic rank.
• Per-study DA + cross-study inverse-variance pooling with random- or fixed-effects model.
• Cochran's I² heterogeneity diagnostics.
• Diagnostic combined-PCoA to spot study effects.
• Method-comparison sweep for meta-analysis (wilcoxon / deseq2 / ancombc).

Outside scope — separate skill:

• Building the per-study AnnData from FASTQs — see omicverse-microbiome-16s-amplicon-dada2.
• Single-cohort DA method comparison — see omicverse-microbiome-da-comparison.
• Phylogenetic harmonisation across studies (different reference trees) — see omicverse-microbiome-phylogeny for the per-study tree, but no cross-study tree alignment is implemented.
• Batch correction in the canonical sense (ComBat / Harmony) — combine_studies only zero-fills and tags obs['study']. For batch correction across cohorts, consider modelling study as a covariate in a per-feature mixed model (out of scope for ov.micro).
• Strain-level meta-analysis — current implementation collapses to a taxonomic rank; species-level cross-cohort matching needs uniform DBs (out of scope).

Branch Selection

combine_studies(rank=...)

• rank='genus' (default) — robust across cohorts, the canonical reporting rank for 16S meta-analyses (Nearing 2022, Pasolli 2017). Use this unless you have a specific reason.
• rank='family' — when even genera don't align well across DBs.
• rank=None — ASV-level; only useful if every cohort was processed against the same DB version with the same denoiser; rare in practice.

meta_da(method=...)

• 'deseq2' — default. Per-study NB-GLM, then inverse-variance pooling on the per-study LFCs. Best calibrated when n>=10/group per cohort.
• 'wilcoxon' — non-parametric per-study, then pool. Use when one cohort has very small n where NB assumptions break.
• 'ancombc' — compositional bias-corrected per-study, then pool. The most rigorous on compositional data but slowest; flag for reportable results.

combine='random_effects' vs 'fixed_effects'

• random_effects (DerSimonian-Laird, default): models a between-study variance component τ² in addition to per-study sampling variance. Conservative when studies disagree (high I²); reduces to fixed-effects when studies agree.
• fixed_effects: assumes one true effect across studies; tighter CIs but mis-leads when studies actually differ. Use only when between-study heterogeneity is provably zero (rare for 16S).
• Default to random-effects for 16S meta-analyses — different cohorts have different recruitment, geography, sample handling.

Cochran's I² interpretation

• I² < 0.25: low heterogeneity — combined effect is well-supported.
• 0.25 ≤ I² < 0.5: moderate; report combined effect but flag.
• 0.5 ≤ I² < 0.75: substantial; combined effect is suspect — does the contrast actually hold across cohorts?
• I² >= 0.75: high; almost certainly a method or covariate problem (or genuine biology that varies across cohorts) — interpret per-study, don't pool.

min_prevalence in meta_da is applied per-study (not after combination), so a feature must clear min_prevalence in at least one study to be tested. Setting it lower (0.05) tests more rare features but inflates FDR-correction burden.

Input Contract

• Per-study AnnDatas all have:
  • obs[group_key] populated with categorical values; the two contrast levels (group_a, group_b) appear in every study (otherwise that study contributes nothing to the contrast).
  • var[rank] for whatever taxonomic rank combine_studies will collapse to. Empty strings (unassigned features) are dropped during collapse.
  • Integer counts in .X (DESeq2 mode) or counts that survive relative=True rescaling (Wilcoxon mode).
• Sample counts per study can be unequal — the random-effects model handles this; just don't have any cohort with n<5/group.
• Different per-study DBs are OK at the genus level (genus names are largely DB-portable). Below genus, alignment breaks.

Minimal Execution Patterns

import omicverse as ov
import anndata as ad
import matplotlib.pyplot as plt
import numpy as np

ov.plot_set()

# 1) Load per-study AnnDatas
adata_A = ad.read_h5ad('cohort_A_genus.h5ad')
adata_B = ad.read_h5ad('cohort_B_genus.h5ad')
adata_C = ad.read_h5ad('cohort_C_genus.h5ad')

# 2) Combine — zero-fills cohort-specific features, tags obs['study']
adata_all = ov.micro.combine_studies(
    [adata_A, adata_B, adata_C],
    study_names=['cohort_A', 'cohort_B', 'cohort_C'],
    rank='genus',
)
print('combined:', adata_all.shape)
print(adata_all.obs['study'].value_counts())

# 3) Diagnostic PCoA on the combined object
ov.micro.Beta(adata_all).run(metric='braycurtis', rarefy=False)
coords = np.asarray(ov.micro.Ordinate(adata_all, dist_key='braycurtis').pcoa(n=2))

# 4) Meta-analysis
meta = ov.micro.meta_da(
    [adata_A, adata_B, adata_C],
    study_names=['cohort_A', 'cohort_B', 'cohort_C'],
    group_key='disease', group_a='CTRL', group_b='CASE',
    method='deseq2',
    rank='genus',
    min_prevalence=0.1,
    combine='random_effects',
)

print(f'features tested cross-cohort: {len(meta)}')
print(f'significant at FDR 0.05    : {(meta["fdr_bh"] < 0.05).sum()}')
print(meta[['feature', 'combined_lfc', 'combined_se', 'z',
            'p_value', 'fdr_bh', 'n_studies', 'I2']].head(10))

# Forest-style top hits with 95 % CI (1.96 × combined_se)
top = meta.head(12).copy().iloc[::-1]
ci = 1.96 * top['combined_se'].values

fig, ax = plt.subplots(figsize=(7, 5))
ax.barh(top['feature'], top['combined_lfc'], xerr=ci,
        edgecolor='black', linewidth=0.4)
ax.axvline(0, color='k', lw=0.7)
ax.set_xlabel('combined log2 fold-change (CASE / CTRL)')
plt.tight_layout(); plt.show()

# Heterogeneity scatter — flag high-I² hits
sig = meta[meta['fdr_bh'] < 0.05].copy()
fig, ax = plt.subplots(figsize=(7, 4))
ax.scatter(sig['combined_lfc'], sig['I2'], s=50, edgecolors='k', linewidths=0.4)
ax.axhline(0.25, color='grey', lw=0.5, ls='--')
ax.axhline(0.75, color='grey', lw=0.5, ls='--')
ax.set_xlabel('combined log2FC'); ax.set_ylabel("Cochran's I²")
plt.tight_layout(); plt.show()

# Per-method comparison — which method recovers planted signals first?
results = {}
for method in ['wilcoxon', 'deseq2', 'ancombc']:
    results[method] = ov.micro.meta_da(
        [adata_A, adata_B, adata_C],
        study_names=['cohort_A', 'cohort_B', 'cohort_C'],
        group_key='disease', group_a='CTRL', group_b='CASE',
        method=method, rank='genus', min_prevalence=0.1,
    )
import pandas as pd
summary = pd.DataFrame({
    'n_tested':       {m: len(r) for m, r in results.items()},
    'n_sig_fdr_0.05': {m: int((r['fdr_bh'] < 0.05).sum()) for m, r in results.items()},
})
print(summary)

Validation

• After combine_studies: adata_all.obs['study'].value_counts() matches per-study sample counts; combined feature count is <= sum(per_study_features) (zero-fills shouldn't lose features).
• Diagnostic PCoA: PC1 colour-mapped by study should look at least somewhat banded — if not, you have miraculously homogeneous data, but check that combine_studies collapsed correctly (rank actually shared).
• meta_da returns n_studies per feature — features that only show up in 1–2 studies have weaker pooled inference. Flag rows where n_studies < len(studies).
• I² above 0.75 on a "significant" feature: do not report as a meta-finding. Interpret per-study or note explicitly that the effect is heterogeneous.
• For known-truth validation (planted features in synthetic data, or housekeeping features in real data): they should appear at the top of the per-study DA AND survive meta-analysis with low I² and small p-value. If a planted feature has high I² in meta_da, the synthetic noise wasn't realistic enough.
• Cross-method comparison: for at least 2/3 methods, the planted features should be in the top 3 hits. Methods that completely miss planted features signal a configuration error (wrong contrast, wrong min_prevalence).

Resource Map

• See reference.md for compact copy-paste snippets per stage.
• See references/source-grounding.md for verified combine_studies / meta_da signatures and the inverse-variance + random-effects implementation details.
• For per-study AnnData ingest, see omicverse-microbiome-16s-amplicon-dada2.
• For single-cohort DA method comparison, see omicverse-microbiome-da-comparison.

Examples

• "Combine three cohorts at the genus level, run a random-effects DESeq2 meta-analysis, and report the top-12 features with 95 % CIs and I² heterogeneity."
• "Compare Wilcoxon / DESeq2 / ANCOM-BC at the meta level — which recovers planted features first?"
• "Diagnose whether a 'significant' meta-hit at FDR 0.05 has acceptable cross-cohort agreement (I²)."
• "Run a fixed-effects meta-analysis only when all three studies agree (low I²)."

References

• Tutorial notebook: t_16s_meta_analysis.ipynb — synthetic 3-cohort meta-analysis with planted vs. cohort-specific features.
• Live API verified — see references/source-grounding.md.