Version Compatibility

Reference examples tested with: pandas 2.2+, DESeq2 1.42+, edgeR 4.0+, limma 3.58+, variancePartition / dream 1.32+, somalier 0.2.18+ (CLI), pyensembl 2.3+, anndata 0.10+

Before using code patterns, verify installed versions match. If versions differ:

• R: packageVersion('<pkg>') then ?function_name to verify parameters
• Python: pip show <package> then help(module.function) to check signatures
• CLI: <tool> --version then <tool> --help to confirm flags

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

Metadata Joins

"Align my sample metadata with my count matrix and build a design" -> Reconcile sample identifiers, validate the join, set factor levels explicitly to control fold-change direction, encode the experimental structure (paired, repeated measures, interaction) in the design formula, and detect swaps before downstream DE.

The Single Most Important Modern Insight -- Alphabetical reference levels invert fold changes silently

DESeq2 picks the reference level alphabetically if not told otherwise. With condition = c('Treated', 'Untreated'), T < U, so Treated becomes the reference. The reported log2FoldChange is then Untreated vs Treated -- the opposite of what the methods section says. No error; the volcano plot looks plausible; the gene list is correct but with reversed sign.

dds$condition <- relevel(dds$condition, ref = 'Untreated')
coldata$condition <- factor(coldata$condition, levels = c('Untreated', 'Treated'))

Set BEFORE DESeq(); relevel-then-DESeq again to take effect.

A second insight: in interaction designs ~ genotype * treatment, results(dds, name='treatment_drug_vs_vehicle') returns the drug effect IN THE WT REFERENCE only, not the marginal/average effect. The interaction coefficient genotypeKO.treatmentdrug is the DIFFERENCE in drug effect between KO and WT (the difference of differences), NOT the drug effect in KO. The cleanest fix is to use ~ 0 + group with group = paste(genotype, treatment, sep='_') so every comparison of interest is a single contrast.

Algorithmic Taxonomy

Pattern	Encoding	Tests	Caveat
Simple two-group	~ condition	results(name='condition_treated_vs_control')	Set reference level explicitly
With known batch	~ batch + condition	results(name='condition_...')	Variable of interest LAST is the convention; not required
Paired (pre/post per subject)	~ subject + condition	results(name='condition_...')	Pairing FIRST; subject absorbs baseline variability
Repeated measures (>2 time per subject)	DREAM `~ condition + (1	subject)`	topTable(coef='condition')
Interaction (2x2)	~ A * B (expands to A + B + A:B)	results(name='A.B') for diff-in-diff; combined factor for cleaner contrasts	resultsNames trap
Multi-group all pairwise	~ 0 + group + makeContrasts	Contrasts named directly	DESeq2 needs intercept; works for edgeR/limma
Multi-level "any change"	LRT reduced = ~ 1	results(dds) from LRT fit	padj is omnibus; LFC is one specific level

Decision Tree by Scenario

Scenario	Recommended approach
Tumor / normal paired by patient	~ patient + tissue; pairing FIRST -- absorbs subject variability
Pre / post drug, same patient (2 time points)	~ subject + condition
Longitudinal, 4+ time points per subject	DREAM mixed model with (1 | subject) random effect
Known batch (sequencing run, library prep date)	~ batch + condition; do NOT subtract
Many batches (>10 levels, n=20-40)	~ condition + (1 | batch) via DREAM; or aggregate batches
Continuous covariate (age, RIN)	Center first; include linearly OR via ns(x, df=3) if non-linear
Mixed-sex cohort	Include sex unless sex-specific; report sex-stratified sensitivity
Multi-group, want pairwise contrasts	~ 0 + group + makeContrasts
Interaction question (does effect of A differ across B?)	Either ~ A * B (handle resultsNames trap) or combined factor ~ 0 + group
Many technical reps within bio reps	duplicateCorrelation (limma) OR aggregate via collapseReplicates (DESeq2)
Cohort >=20 samples	Run somalier or NGSCheckMate genotype check at the matrix-build step

Basic Join

Goal: Align count matrix columns with metadata rows; remove samples present in only one source; verify alignment before downstream use.

Approach: Intersect sample identifiers; reorder both data sources; assert match.

import pandas as pd

counts = pd.read_csv('counts.tsv', sep='\t', index_col=0)
metadata = pd.read_csv('metadata.csv', index_col=0)

common = counts.columns.intersection(metadata.index)
only_counts = set(counts.columns) - set(metadata.index)
only_meta = set(metadata.index) - set(counts.columns)
if only_counts: print(f'In counts not metadata: {only_counts}')
if only_meta: print(f'In metadata not counts: {only_meta}')

counts = counts[common]
metadata = metadata.loc[common]
assert all(counts.columns == metadata.index)

common <- intersect(colnames(counts), rownames(coldata))
counts <- counts[, common]
coldata <- coldata[common, , drop = FALSE]
stopifnot(all(colnames(counts) == rownames(coldata)))

When sample names differ by formatting (underscore vs dash, BAM suffix, case), try systematic transformations -- replace _ <-> -, strip .bam, lower-case, take prefix before _ -- before giving up.

Reference Level (Critical -- Set Before DESeq)

Goal: Pick the baseline against which fold changes are reported, controlling sign direction.

Approach: relevel() or construct the factor with explicit levels=. Set BEFORE DESeq() runs.

coldata$condition <- factor(coldata$condition, levels = c('control', 'treated'))

coldata$condition <- relevel(coldata$condition, ref = 'control')

metadata['condition'] = pd.Categorical(metadata['condition'],
                                        categories=['control', 'treated'],
                                        ordered=True)

With factor levels explicit, the LFC reads treated / control -- treated up means LFC > 0.

A reminder for edgeR / limma users: makeContrasts(Treated - Control) is explicit and immune to the reference-level trap. Same caution applies less because the user names the contrast.

Paired Designs

design = ~ patient + tissue

Pairing variable FIRST (convention). Patient absorbs inter-subject baseline variability, dramatically increasing power for the tissue effect.

For DESeq2:

dds <- DESeqDataSetFromMatrix(counts, coldata, design = ~ patient + tissue)
dds <- DESeq(dds)
res <- results(dds, name = 'tissue_tumor_vs_normal')

For edgeR:

design <- model.matrix(~ patient + tissue, coldata)
y <- estimateDisp(y, design, robust = TRUE)
fit <- glmQLFit(y, design, robust = TRUE)
qlf <- glmQLFTest(fit, coef = 'tissuetumor')

Common mistake: writing ~ tissue + patient. Numerically the model is the same; the convention of pairing-first improves readability and matches the natural mental model.

Interaction Terms -- the resultsNames Trap

design = ~ genotype + treatment + genotype:treatment
dds <- DESeqDataSetFromMatrix(counts, coldata, design = design)
dds <- DESeq(dds)
resultsNames(dds)

Output names (after relevel):

"Intercept"
"genotype_KO_vs_WT"
"treatment_drug_vs_vehicle"
"genotypeKO.treatmentdrug"

Question	Wrong answer	Right answer
Drug effect averaged over genotypes	results(name='treatment_drug_vs_vehicle') -- this is drug effect in WT only	Combined factor ~ 0 + group; or contrast that explicitly averages
Is drug effect different between genotypes?	n/a	results(name='genotypeKO.treatmentdrug') -- the interaction coefficient IS the difference of differences
Drug effect in KO	results(name='treatment_drug_vs_vehicle') -- WRONG, this is drug in WT	results(contrast=list(c('treatment_drug_vs_vehicle', 'genotypeKO.treatmentdrug'))) (sum of main + interaction)

The cleaner alternative for designs with many contrasts of interest:

coldata$group <- factor(paste(coldata$genotype, coldata$treatment, sep = '_'))
dds <- DESeqDataSetFromMatrix(counts, coldata, design = ~ 0 + group)
dds <- DESeq(dds)

res_drug_in_ko <- results(dds, contrast = c('group', 'KO_drug', 'KO_vehicle'))
res_drug_in_wt <- results(dds, contrast = c('group', 'WT_drug', 'WT_vehicle'))
res_diff       <- results(dds, contrast = list(c('groupKO_drug', 'groupWT_vehicle'),
                                                c('groupKO_vehicle', 'groupWT_drug')))

~ 0 + group parameterization is the long-standing edgeR / limma recommendation (Smyth and Robinson User's Guides) for any design with multiple pairwise contrasts of interest.

LRT and the Reduced Model

dds <- DESeq(dds, test = 'LRT', reduced = ~ batch)

Reduced model drops the term being tested. With design = ~ batch + condition and reduced = ~ batch, the LRT tests condition. With interaction designs:

dds <- DESeq(dds, test = 'LRT', reduced = ~ genotype + treatment)

(Tests the interaction.)

The reduced model must be NESTED in the full model (every term in reduced must appear in full).

Continuous Covariates

Encoding	Assumption	When
Linear (+ age)	log-expression linear in age	Limited range, biologically linear
Centered linear (+ I(age - mean(age)))	As linear, interpretable intercept	Standard for age-RIN-day covariates
Natural spline (+ ns(age, df=3))	Smooth nonlinear	Wide age range with non-monotonic effects
Polynomial (+ poly(age, 2))	Quadratic; orthogonal polynomials	Limited use; splines usually better

coldata$age_c <- coldata$age - mean(coldata$age)
design = ~ age_c + RIN + condition

library(splines)
design = ~ ns(age, df = 3) + condition

DO NOT include library size as a covariate -- it is handled by size factors / normalization factors internally.

Repeated Measures -- duplicateCorrelation vs dream

Goal: Correctly model within-subject correlation when the same subject contributes multiple samples.

Approach: For technical reps within bio reps OR paired pre/post: duplicateCorrelation (limma) is adequate. For >2 time points per subject or random slopes: DREAM (variancePartition).

library(limma)
library(edgeR)

v <- voom(y, design)
corfit <- duplicateCorrelation(v, design, block = coldata$donor)
v <- voom(y, design, block = coldata$donor, correlation = corfit$consensus)
corfit <- duplicateCorrelation(v, design, block = coldata$donor)
fit <- lmFit(v, design, block = coldata$donor, correlation = corfit$consensus)
fit <- eBayes(fit, robust = TRUE)

The double pass is intentional: estimate correlation, re-voom with correlation, re-estimate, fit. Limma's duplicateCorrelation assumes ONE within-subject correlation across all genes -- approximation.

For proper per-gene mixed models:

library(variancePartition)

form <- ~ condition + (1 | donor)
vobj <- voomWithDreamWeights(y, form, coldata)
fitmm <- dream(vobj, form, coldata)
fitmm <- eBayes(fitmm)
tt <- topTable(fitmm, coef = 'condition')

See differential-expression/timeseries-de for full longitudinal designs.

Before committing to a design, variancePartition::fitExtractVarPartModel(vobj, form, coldata) quantifies the fraction of expression variance explained by each covariate, gene-by-gene. If a candidate "nuisance" covariate explains <1% of variance across most genes, it can usually be dropped; if a known biological factor explains <5% and isn't of direct interest, model it as a random effect rather than fixed.

Sample Swap Detection (Mandatory for Cohort >= 20)

Sex check via XIST and chrY expression

Goal: Detect mislabeled samples by checking that gene-expression sex matches reported sex.

Approach: Compare XIST expression (high in XX, low/absent in XY) against chrY-gene expression (DDX3Y, RPS4Y1, UTY, KDM5D, EIF1AY -- high in XY, absent in XX).

import pandas as pd

def sex_check(counts, metadata, sex_column='sex'):
    y_genes = ['DDX3Y', 'RPS4Y1', 'UTY', 'KDM5D', 'EIF1AY']
    y_avail = [g for g in y_genes if g in counts.index]
    if 'XIST' not in counts.index or not y_avail:
        return None
    predicted = pd.Series('unknown', index=counts.columns)
    predicted[counts.loc[y_avail].sum() > counts.loc['XIST']] = 'M'
    predicted[counts.loc['XIST'] > counts.loc[y_avail].sum()] = 'F'
    if sex_column in metadata.columns:
        mis = predicted != metadata[sex_column]
        if mis.any():
            print(f'SEX MISMATCHES: {list(metadata.index[mis])}')
    return predicted

sex_check <- function(counts, coldata, sex_col = 'sex') {
    y_genes <- c('DDX3Y', 'RPS4Y1', 'UTY', 'KDM5D', 'EIF1AY')
    y_expr <- colSums(counts[intersect(y_genes, rownames(counts)), , drop = FALSE])
    predicted <- ifelse(y_expr > counts['XIST', ], 'M', 'F')
    mis <- predicted != coldata[[sex_col]]
    if (any(mis)) cat('Sex mismatches:', colnames(counts)[mis], '\n')
    predicted
}

CAVEAT: tumors with X loss, sex chromosome aneuploidies, HeLa (XXX with mixed inactivation) muddy this. Genotype-based methods are more robust.

Genotype-based: somalier and NGSCheckMate

somalier extract -d extracted/ --sites sites.GRCh38.vcf.gz \
    -f reference.fa sample.bam
somalier relate --infer extracted/*.somalier

ncm_fastq.py -l fastq_list.txt -O outdir -bed common_sites.bed

Somalier (Pedersen et al. 2020 Genome Med 12:62) extracts a few thousand SNP sketches per sample (sub-second per sample) and computes pairwise relatedness from BAM/CRAM/VCF. NGSCheckMate (Lee et al. 2017 NAR 45:e103) computes VAF correlation across a common-SNP panel; works on FASTQ/BAM/VCF including RNA-seq.

For any cohort >=20 samples, run one of these at the matrix-build step. Catching a swap in raw data is cheap; finding it after DE is expensive.

SABV -- Sex as Biological Variable

NIH 2016+ requires sex consideration in vertebrate animal and human studies. Mauvais-Jarvis F et al. 2020 Lancet 396:565 reviews effect-size differences across diseases.

Practical implication:

• Always include sex in the metadata, even when not in the model.
• For sex-balanced cohorts, ~ sex + condition rarely hurts and captures real biology.
• For sex-confounded cohorts (all-male disease cohort vs mixed-sex control), can't rescue but documents the limitation.
• For chrX/chrY analyses, sex MUST be in the model OR the analysis is uninterpretable.
• Report DE counts overall AND sex-stratified.

Simpson's Paradox -- Collapsing Technical Replicates

Goal: Aggregate technical replicates from the same subject correctly; never treat them as independent biological replicates.

Approach: Sum (not average) technical replicates of the same subject BEFORE downstream DE.

library(DESeq2)
dds_collapsed <- collapseReplicates(dds, groupby = dds$subject)

counts_per_subject = counts.T.groupby(metadata['subject']).sum().T
metadata_per_subject = metadata.drop_duplicates(subset='subject').set_index('subject')

Why sum and not average? Reads add. Two technical replicates yielding 1M reads each are equivalent to one library yielding 2M reads. Averaging would understate the effective library size.

Treating technical replicates as independent biological samples is the cardinal sin: it inflates the apparent sample size and deflates standard errors. With 4 patients x 3 tech reps = 12 samples, naive DE assumes 12 independent observations; the truth is closer to 4. p-values are compressed ~3x.

High-Cardinality Categorical Covariates

Goal: Handle batch / lane / well covariates with many levels without making the design matrix singular.

Approach: Aggregate to fewer levels, model as random effect via DREAM, or drop if confounded.

ct <- table(coldata$condition, coldata$batch)
ct

ad <- alias(model.matrix(~ batch + condition, coldata))
ad$Complete

For a batch with 30 levels in n=40 samples: 30 batch coefficients + condition + intercept = 32 parameters for 40 observations. Symptoms: Matrix not positive definite (DESeq2), degenerate p-values (limma).

Fixes:

• Aggregate batches (sequencing run, sequencing pool, library prep date as proxy).
• Random effect: ~ condition + (1 | batch) via DREAM borrows information across batch levels via shrinkage.
• Drop the covariate if confounded with condition (alias() reveals collinearity).

~ 0 + group Parameterization

design_default <- model.matrix(~ group, coldata)
# columns: (Intercept), groupB, groupC  -- A is reference

design_nointercept <- model.matrix(~ 0 + group, coldata)
# columns: groupA, groupB, groupC  -- each column is mean of that group

With ~ 0 + group, every contrast reads as B - A:

library(limma)

con <- makeContrasts(BvsA = groupB - groupA,
                     CvsA = groupC - groupA,
                     BvsC = groupB - groupC,
                     levels = design_nointercept)
fit <- glmQLFit(y, design_nointercept, robust = TRUE)
qlf <- glmQLFTest(fit, contrast = con[, 'BvsA'])

DESeq2 needs an intercept internally, so ~ 0 + group works directly with edgeR/limma but DESeq2 uses contrast= to achieve the same effect.

Create DESeq2 / edgeR / AnnData Containers

dds <- DESeqDataSetFromMatrix(as.matrix(counts), coldata, design = ~ batch + condition)

y <- DGEList(counts = as.matrix(counts), group = coldata$condition)
y$samples <- cbind(y$samples, coldata)

adata <- ad.AnnData(X = t(as.matrix(counts)), obs = coldata, var = data.frame(row.names = rownames(counts)))

AnnData convention is cells (samples) in rows -- transpose from the typical R genes-in-rows convention.

Per-Method Failure Modes

Fold-change direction reversed

Trigger: Methods says "treated vs control"; published volcano shows expected up-genes on the LEFT.

Mechanism: Factor levels left at alphabetical default; c('Treated','Untreated') -> T < U -> Treated is reference -> LFC is Untreated/Treated.

Symptom: Known up-regulated genes appear down; reviewer questions direction.

Fix: relevel(coldata$condition, ref = 'Untreated') BEFORE DESeq(). Re-run.

Interaction coefficient mistaken for main effect

Trigger: ~ A * B design; results(name='B_drug_vs_vehicle') reported as "drug effect"; reviewer asks about genotype-specific effect.

Mechanism: With interaction, B_drug_vs_vehicle is drug effect IN THE A REFERENCE LEVEL only, not averaged across A.

Symptom: Drug effect doesn't match the marginal estimate from a separate ~ B-only fit.

Fix: Use ~ 0 + group with combined factor; OR extract per-stratum results explicitly using contrasts that sum main + interaction.

Pseudoreplication -- 12 samples, only 3 subjects

Trigger: 3 subjects x 4 conditions = 12 samples; vanilla DESeq2 with ~ condition; many DE genes.

Mechanism: Same subject contributes multiple observations; not independent. Effective sample size for testing condition is ~3, not 12.

Symptom: p-value histogram anti-conservative; replication low.

Fix: Include subject in design (~ subject + condition) OR use DREAM with random subject.

Sample swap caught in DE results, not metadata

Trigger: PCA shows "control" sample clustering with treated; investigation reveals it was mislabeled at thaw.

Mechanism: Manual sample tracking is error-prone; cohort >=20 inevitably has swaps.

Symptom: One sample dramatically off its group cluster; DE gene list dominated by sample-specific effects.

Fix: Run somalier or NGSCheckMate at the matrix-build step, BEFORE DE. Catching a swap early is cheap; finding it post-DE is expensive.

Sex confounded, chrY genes dominate top hits

Trigger: Mixed-sex cohort; sex not in design; PCA shows clear sex split on PC1.

Mechanism: Sex distribution differs between groups; "treatment effect" partially captures sex.

Symptom: Top DE genes are DDX3Y, RPS4Y1, UTY (chrY) and XIST -- not biology of interest.

Fix: Add sex to design (~ sex + condition). For sex-specific analyses, stratify and report each separately.

duplicateCorrelation single-pass

Trigger: limma user wrote a one-pass duplicateCorrelation + lmFit; QC reviewer asks why no re-voom.

Mechanism: The proper pattern is: voom -> dupCor -> re-voom WITH correlation -> dupCor again -> lmFit. The first voom doesn't know about block structure; re-voom with correlation gets better weights.

Symptom: Slightly inflated DE counts vs the two-pass pattern.

Fix: Implement the two-pass pattern per the limma User's Guide (section 9.7).

Common errors

Error / symptom	Cause	Fix
Sample names don't match between counts and metadata	Underscore/dash inconsistency, BAM suffix, case	fuzzy_match_samples() or manual normalization; report what failed
DESeq2 design not full rank	Confounded covariates	alias(design)$Complete to identify; aggregate or drop
Matrix not positive definite	High-cardinality batch with few samples	Aggregate batches or use random effect via DREAM
LFC direction reversed	Alphabetical reference level	relevel() before DESeq()
results(name='...') returns drug effect in WT only	Interaction design and naming trap	Use combined factor ~ 0 + group; or contrast summing main + interaction
Inflated DE list with 12 samples from 3 subjects	Pseudoreplication	Include subject; or use DREAM
Sex effect appears as treatment effect	Sex not in design	Add ~ sex + condition
Sample distance heatmap shows mixing groups	Likely swap	Run somalier or NGSCheckMate

References

• Love MI, Huber W, Anders S. 2014. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15(12):550. doi:10.1186/s13059-014-0550-8
• Robinson MD, McCarthy DJ, Smyth GK. 2010. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26(1):139-140. doi:10.1093/bioinformatics/btp616
• Ritchie ME et al. 2015. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res 43(7):e47. doi:10.1093/nar/gkv007
• Smyth GK, Michaud J, Scott HS. 2005. Use of within-array replicate spots for assessing differential expression in microarray experiments. Bioinformatics 21(9):2067-2075. doi:10.1093/bioinformatics/bti270
• Hoffman GE, Roussos P. 2021. dream: powerful differential expression analysis for repeated measures designs. Bioinformatics 37(2):192-201. doi:10.1093/bioinformatics/btaa687
• Hoffman GE, Schadt EE. 2016. variancePartition: interpreting drivers of variation in complex gene expression studies. BMC Bioinformatics 17:483. doi:10.1186/s12859-016-1323-z
• Pedersen BS, Bhetariya PJ, Brown J, Kravitz SN, Marth GT, Jensen RL, Bronner MP, Underhill HR, Quinlan AR. 2020. Somalier: rapid relatedness estimation for cancer and germline studies using efficient genome sketches. Genome Medicine 12:62. doi:10.1186/s13073-020-00761-2
• Lee S, Lee S, Ouellette S, Park W-Y, Lee EA, Park PJ. 2017. NGSCheckMate: software for validating sample identity in next-generation sequencing studies within and across data types. Nucleic Acids Res 45(11):e103. doi:10.1093/nar/gkx193
• Mauvais-Jarvis F, Bairey Merz N, Barnes PJ et al. 2020. Sex and gender: modifiers of health, disease, and medicine. Lancet 396(10250):565-582. doi:10.1016/S0140-6736(20)31561-0

Related Skills

• counts-ingest - Building count matrices before metadata join
• gene-id-mapping - Annotating result tables with symbols
• normalization - Reference for downstream normalization choice
• sparse-handling - AnnData obs metadata convention
• differential-expression/deseq2-basics - Where the design formula matters; relevel; interactions
• differential-expression/edger-basics - edgeR design matrix conventions
• differential-expression/batch-correction - Design-inclusion vs subtraction; confounding
• differential-expression/timeseries-de - Repeated measures; DREAM mixed model details
• single-cell/preprocessing - scRNA-seq sample metadata handling