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Defines cell populations in flow and spectral cytometry through manual gates (rectangle, polygon, quadrant, boolean) and reproducible automated gating (openCyto gating templates, flowDensity data-driven thresholds, flowClust model-based gates), organized as a hierarchical GatingSet (flowWorkspace) and round-tripped with FlowJo via CytoML. Covers the canonical gate order (time -> debris -> singlets -> live -> lineage), FMO-vs-isotype boundary setting, gate-order dependence and recompute semantics, rare-event/MRD gating, and per-population statistics. Use when building a gating strategy, automating a manual FlowJo scheme across samples, choosing manual vs data-driven gates, or extracting population frequencies.

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Assesses the quality and completeness of a genome annotation with BUSCO (conserved single-copy ortholog recovery), OMArk (proteome completeness, consistency, and contamination), CheckM2 (prokaryotic completeness/contamination), and a gene-set sanity panel (gene count, mono-exonic fraction, protein-length distribution, mRNA:gene ratio, coding density). Covers the assembly-BUSCO-vs-proteome-BUSCO diagnostic, what BUSCO-Duplicated really means, why gene count is a vanity metric, and the QC of transferred annotations. Use when judging whether an annotation is good enough to publish or submit, diagnosing a suspect annotation, or comparing annotation completeness across pipelines.

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Infer transcription factor regulons from single-cell RNA-seq with pySCENIC by combining GRNBoost2 co-expression, cisTarget motif-enrichment pruning, and AUCell per-cell activity scoring. Covers the motif-pruning-as-directionality principle, regulon specificity scoring, run-to-run stability, and database/species matching. Use when identifying TF regulons, scoring TF activity per cell, finding master regulators of cell identity, or comparing regulon activity across conditions. For enhancer-driven multiomic GRNs see multiomics-grn; for bulk inference and VIPER protein-activity see grn-inference.

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Aligns and QCs methylated-RNA-immunoprecipitation (MeRIP / m6A-seq) IP and input libraries using STAR or HISAT2 splice-aware mapping, samtools sort/index, IP/input matched-pair tracking, antibody-lot metadata recording, replicate concordance via deepTools multiBamSummary + plotCorrelation, IP enrichment QC via plotFingerprint and per-transcript IP/input ratio distributions, library-complexity saturation curves via PreSeq c_curve / lc_extrap, and the explicit do-NOT-deduplicate convention for standard non-UMI MeRIP. Use when preparing paired IP and input BAM files for exomePeak2 / MeTPeak / MACS3 peak calling, evaluating MeRIP replicate concordance and IP enrichment, deciding whether to deduplicate (standard MeRIP typically NOT — see Tips), choosing genome-vs-transcriptome alignment for downstream peak vs m6Anet workflows, recording antibody clone and lot metadata for downstream cross-batch reconciliation, detecting failed IPs via saturation curves and IP/input distribution shape, or generating IP-over-Input bigWig tracks for downstream visualisation.

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Visualises RNA-modification data with transcript-feature metagene plots (Guitar GuitarPlot with 5'UTR / CDS / 3'UTR scaling; MetaPlotR; deepTools `computeMatrix scale-regions`), peak-centred heatmaps (ComplexHeatmap; deepTools plotHeatmap), IP-vs-input paired browser tracks (bigWig of log2 IP/input via deepTools `bamCompare`; ggcoverage; pyGenomeTracks; Gviz; IGV / UCSC track hubs), DRACH sequence-logo plots (ggseqlogo; MEME), 5'UTR / CDS / 3'UTR stacked-bar feature-distribution summaries, and volcano / MA plots for differential modification. Establishes stop-codon enrichment in the metagene plot as the biological QC anchor for any MeRIP dataset (Dominissini 2012 *Nature* 485:201; Meyer 2012 *Cell* 149:1635 — concurrent founding papers from different labs both showed this independently). Use when producing the canonical metagene plot with stop-codon enrichment as a QC anchor, building paired IP/input genome-browser tracks at single-locus resolution, plotting peak-centred heatmaps clustered by condition, summarising peak distribution across transcript features for figure 1, generating DRACH motif logos as sanity checks on the peak set, rendering volcano plots of differential m6A, or reproducing the Dominissini 2012 / Meyer 2012 stop-codon enrichment plot.

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Stores and operates on sparse expression matrices for single-cell and large bulk RNA-seq, covering dgCMatrix/dgRMatrix/dgTMatrix when-each-is-fast, the dgCMatrix (CSC, R) <-> CSR (Python) implicit transpose, AnnData (cells-rows) <-> SingleCellExperiment (cells-cols) orientation flip, HDF5/h5ad vs Zarr cloud-native shift, HDF5SummarizedExperiment + DelayedArray for out-of-memory bulk, scanpy backed mode for large h5ad, the ~10-15% density crossover where dense beats sparse, 10X format proliferation (MTX vs CellRanger H5 vs h5ad), the dense-conversion memory blow-up, and Dask + Zarr for consortium-scale matrices. Use when choosing sparse format, working with single-cell-sized matrices, importing/exporting 10X, debugging R/Python interop transposes, processing matrices too large for RAM, or building cloud-native pipelines.

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Quantifies biodiversity from species abundance/incidence tables using Hill numbers (iNEXT) with coverage-based rarefaction-extrapolation (Chao & Jost 2012), asymptotic richness via Chao1/ACE/jackknife as a lower bound, Baselga turnover/nestedness partition with the Podani alternative as sensitivity check, mandatory Hellinger transformation before ordination (Legendre & Gallagher 2001), Faith PD and SES_MPD/SES_MNTD with explicit null-model choice, and Maire 2015 functional-diversity dimensionality optimization. Use when comparing diversity across sites with unequal sampling effort, picking the right richness estimator for singleton-heavy amplicon data, partitioning beta diversity into turnover vs nestedness, reporting Hill-number effective species counts rather than raw entropies, computing SES_MPD with explicit null-model justification, or deciding whether to apply standard metrics to compositional amplicon data. Not for clinical 16S microbiome diversity (see microbiome/diversity-analysis).

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Detects acquired antimicrobial-resistance determinants and chromosomal point-mutation resistance in bacterial assemblies using AMRFinderPlus, ResFinder 4.0 (acquired + PointFinder), CARD-RGI, abritAMR, staramr, and species-specific callers (TB-Profiler, Mykrobe). Harmonises cross-tool output via hAMRonization, contextualises determinants with mobile-genetic-element annotation (MOB-suite, PlasmidFinder, MobileElementFinder, ICEberg), predicts phenotype against EUCAST or CLSI breakpoints, and translates calls into WHO GLASS reporting categories. Use when screening clinical or surveillance isolates for AMR, distinguishing acquired vs intrinsic vs point-mutation resistance, calling rpoB / katG / pncA / gyrA / mgrB mutations, reconciling AMRFinderPlus vs RGI vs ResFinder disagreement, contextualising carbapenemases or mcr alleles on plasmids, predicting susceptibility from genotype against the WHO Mtb 2nd-edition catalogue, or building a hAMRonized multi-lab AMR surveillance pipeline.

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Differential abundance (DA) and differential state (DS) analysis for flow and mass cytometry - tests which cell populations change in frequency or marker expression between conditions using diffcyt (edgeR/voom/GLMM for DA, limma/LMM for DS), with cydar, CITRUS, and compositional methods (sccomp, scCODA, DCATS) as alternatives. Covers the sample-is-the-experimental-unit principle, design/contrast and mixed-model formulas, compositionality of cluster proportions, and FDR across clusters. Use when comparing populations between groups, choosing a DA method, handling paired/batch designs, or deciding whether compositional correction is needed.

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Identifies differential m6A methylation between conditions from MeRIP-seq paired IP/input data using exomePeak2 with `bam_ip` + `bam_input` (control arm) and `bam_treated_ip` + `bam_treated_input` (treatment arm) for integrated GC-bias-aware differential calling (Liu 2022 *NAR Genom Bioinform* 4:lqac046), QNB beta-binomial test (Liu 2017 *BMC Bioinformatics* 18:387), MeTDiff HMM-based differential bundled with MeTPeak, RADAR (Zhang 2019 *Genome Biol* 20:294) with its `filterBins -> diffIP -> reportResult` workflow, and the defensible paired-symmetric case of edgeR / DESeq2 on featureCounts-on-peaks matrices when batch / lot covariates need explicit fixed-effect handling (exomePeak2's top-level API does NOT accept arbitrary covariates). Covers paired vs unpaired vs interaction designs, batch confounding (antibody lot, RNA prep, sequencing run) and the per-lot meta-analysis strategy when exomePeak2 is the primary caller, the stoichiometry-vs-expression-vs-IP-efficiency confound that all MeRIP differential methods inherit, normalisation choice (size factor on IP, on input, on per-sample IP/input ratio), the McIntyre 2020 reproducibility caveat, effect-size filtering as a guardrail against under-powered N=2 designs, and orthogonal-validation routes for absolute stoichiometry (GLORI / SAC-seq / m6Anet `mod_ratio`). Use when comparing m6A levels across two or more conditions, choosing between exomePeak2 / QNB / RADAR / MeTDiff for a given design, handling batch confounding when exomePeak2's API is too rigid, normalising against input properly, distinguishing real hyper- / hypo-methylation from expression-level shifts, applying effect-size thresholds, interpreting volcano plots of differential peaks, or planning a follow-up orthogonal stoichiometry validation.

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Assigns isolate identity at the right resolution for the question -- ANI / Mash species triage, 7-locus MLST historical comparability, cgMLST / wgMLST outbreak resolution (chewBBACA, BIGSdb, Ridom SeqSphere, EnteroBase HierCC), in-silico serotyping (SISTR, SeqSero2 for Salmonella; SerotypeFinder for E. coli; Kaptive K/O for Klebsiella; SeroBA for pneumococcus; spa + SCCmec for S. aureus), and lineage callers (TB-Profiler / Mykrobe Coll-Napier barcode for MTBC, Pangolin + Nextclade for SARS-CoV-2, PopPUNK GPSC for S. pneumoniae). Use when typing bacterial isolates for surveillance or outbreak investigation, choosing between cgMLST allele distance and core-SNP distance for cluster definition, harmonising calls across schemas / database versions (chewBBACA vs Ridom vs EnteroBase), assigning MTBC lineage with the Napier 2020 90-SNP barcode, calling Salmonella serovar via SISTR with monophasic Typhimurium awareness, running Pangolin UShER mode with explicit pangolin-data version pinning, or selecting a typing resolution to match the surveillance question.

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Controls error rates across thousands of simultaneous tests in genomics discovery using false-discovery-rate methods (Benjamini-Hochberg 1995; Benjamini-Yekutieli 2001 for arbitrary dependence; Storey q-value with pi0 estimation; local FDR; independent filtering Bourgon 2010; covariate-weighted FDR via IHW Ignatiadis 2016), plus family-wise error control (Bonferroni, Holm) and the GWAS genome-wide threshold. Covers the FDR-versus-FWER choice as the discovery-versus-confirmatory distinction, the dependence assumptions behind BH (PRDS) versus BY, pi0 estimation, the independent-filtering and false-coverage-rate traps, and reproducibility ranking via IDR (Li 2011). Use when correcting p-values from genome-wide tests, choosing between BH/BY/q-value/Bonferroni, setting an FDR threshold, applying IHW or independent filtering, or interpreting q-values. For confirmatory trials with few pre-specified endpoints (closed testing, graphical/gatekeeping), see clinical-biostatistics/multiplicity-graphical.

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Processes eDNA metabarcoding from raw paired-end reads to species tables, navigating ASV (DADA2, UNOISE3) vs OTU (swarm v2) decision (Callahan 2017 vs Schloss multi-copy-16S critique), marker/primer choice (Leray COI, MiFish 12S, 515F/806R 16S, ITS2) with primer-specific bias, OBITools3 v3 command-name break (obi stats plural; .tar.gz taxonomy), tag-jumping with dual-indexing (Schnell 2015; NovaSeq 10x MiSeq), decontam as screening-not-classifier (Davis 2018), read-counts-not-abundance critique (Lamb 2019), site-occupancy modeling (Ficetola 2015), Naive-Bayes calibration limits (Bokulich 2018), and eDNA decay (Strickler 2015). Use when going from raw eDNA FASTQ to species tables, picking marker + denoising pipeline, deciding whether read counts represent abundance, applying occupancy modeling, configuring OBITools3 v3, or interpreting decontam output. Not for clinical 16S microbiome (see microbiome/amplicon-processing).

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Quantifies CRISPR editing outcomes with CRISPResso2 (Clement 2019 Nat Biotechnol) across Cas9-nuclease (indels, HDR), CBE and ABE base editors (target conversion + bystander), and prime editor (pegRNA-templated) modes. Covers single-amplicon (CRISPResso), multi-sample batch (CRISPRessoBatch), pooled-amplicon (CRISPRessoPooled), WGS off-target (CRISPRessoWGS), and sample-comparison (CRISPRessoCompare) workflows; quantification-window math that controls what is called edited; substitution-vs-indel diagnostic to distinguish BE from Cas9 contamination; MMEJ deletion pattern interpretation; allele-frequency tables; and failure modes from amplicon misalignment or contamination. Use when quantifying editing from amplicon sequencing, choosing CRISPResso mode by design, distinguishing intended edits from bystanders and indel byproducts, debugging low-alignment runs, or generating publication-grade editing reports.

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Find cross-database references between NCBI databases using Biopython Bio.Entrez (ELink). Use when navigating gene to protein/structure, sequence to publication, PubMed to GEO, BioProject to SRA runs, or discovering all link relationships for a record. Covers linkname semantics, cmd= variants, asymmetric link warnings, neighbor_history for >200 input IDs, and per-database link tables.

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Plot per-group distributions of continuous data using boxplots, violins, beeswarms, quasirandom jitter, and raincloud plots with sample-size honesty (Weissgerber 2015), KDE-bandwidth awareness, and N-aware encoding choices. Use when comparing distributions across a small number of groups — expression per cluster, biomarker per arm, scores per condition — and the bar-of-mean default is misleading.

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Build Manhattan, Miami, QQ, and locuszoom-style regional plots from GWAS, TWAS, PWAS, and QTL summary statistics with correct genomic-inflation diagnostics, multi-trait overlays, lead-SNP labeling, and LD-aware regional rendering. Use when visualizing association results across the genome, comparing two traits, computing genomic inflation lambda, or zooming into a locus with LD coloring.

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Designs and analyzes in vivo CRISPR screens in animal tumor models, organoids, and immune-cell adoptive transfers. Covers bottleneck math (250x cells/sgRNA requires ~25M cells implanted; impossible for most syngeneic models, forcing focused libraries), focused library design (Manguso 2017 Nature 547:413 immune screen; Chen 2015 tumor screens), CRISPR-StAR intrinsic-control screening (Uijttewaal 2025 Nat Biotechnol 43:1848), clonal-dynamics-limited detection, tumor-explant DNA recovery, syngeneic vs xenograft vs PDX considerations, and the relationship to downstream MAGeCK / drugZ analysis. Use when designing in vivo CRISPR screens for tumor / immune / metastasis biology, choosing focused vs genome-wide for animal models, addressing bottleneck-induced clonal collapse, picking the syngeneic / xenograft / PDX model, integrating in vivo with in vitro results, or applying CRISPR-StAR for animal experiments.

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Build clustered heatmaps for expression matrices and other features-by-samples data with rigorous distance/linkage/scaling choices, robust color mapping, optimal leaf ordering, and ComplexHeatmap/pheatmap/seaborn rendering. Covers the ward.D vs ward.D2 trap, the row-vs-column scaling decision, multi-track annotations, oncoPrint, and raster rendering for large matrices. Use when visualizing expression patterns across samples or identifying co-regulated clusters.

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Analyzes pooled CRISPR screens with MAGeCK (Li et al 2014), covering count generation (mageck count), the RRA two-condition workflow (mageck test using alpha-RRA over per-sgRNA negative-binomial p-values), the MLE multi-condition workflow (mageck mle with explicit design matrix and beta-score output), normalization choice (median vs total vs control-sgRNA vs spike-in), sgRNA efficiency injection, paired-sample testing, time-course design, drug-screen versus dropout-screen design matrices, MAGeCKFlute and MAGeCK-VISPR downstream visualization, and decision logic for when to use MAGeCK vs JACKS / BAGEL2 / drugZ / Chronos. Use when running a fresh CRISPR screen analysis, picking RRA vs MLE for the experimental design, choosing a normalization method from QC signatures, debugging MLE convergence failure or NaN beta scores, comparing MAGeCK output across tools, or building a batch-aware multi-cell-line / multi-condition MLE design matrix.

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Build circular genome visualizations using circlize (R), pyCirclize (Python), or Circos (Perl CLI) with ideogram tracks, multi-data tracks (scatter, histogram, heatmap), chord/link arcs for interactions, and explicit circos.clear() between plots. Covers when circular is appropriate vs when Cartesian wins (Cleveland-McGill 1984), karyograms, and chromosome adjacency in chord diagrams. Use when adjacency on the circle conveys meaning — chromosome-level overview, structural variants, Hi-C interactions, cross-genome comparisons.

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Pull pre-computed ortholog calls from public databases (OrthoDB, Ensembl Compara, OMA browser, eggNOG, PANTHER, KEGG Orthology, HomoloGene) via their REST APIs. Use when orthologs are already curated upstream, when the question is "what is the X ortholog of Y" rather than "how to infer orthology de novo", when batch-mapping gene IDs across species, or when comparing the resources for consensus calls. Encodes confidence-level semantics, 1:1 vs 1:many vs many:many, HomoloGene deprecation, and when to defect to de novo computation.

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Query the Ensembl REST API for gene/transcript/protein lookup, sequence retrieval, comparative genomics (Compara), variant effect prediction (VEP), regulatory features, and cross-species ortholog/paralog calls. Use when pulling Ensembl-native data (Ensembl Gene IDs, version-pinned releases, archive endpoints for reproducibility), gene/transcript/exon structure with stable IDs, or VEP for variant annotation. Encodes the 15 req/sec rate limit, archive (e110.rest.ensembl.org) for reproducibility, Ensembl divisions (vertebrates / plants / fungi / metazoa / bacteria), and the symbol-vs-ID stability problem.

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Identify recurrent and driver copy number alterations across a tumor cohort with GISTIC2 (G-score, Ziggurat deconstruction, focal vs broad/arm-level analysis, q-values from permutation) and quantify copy-number signatures with the Steele 2022 COSMIC framework and the Drews 2022 CINSignatures framework. Covers driver-gene localization from recurrence peaks, distinguishing focal drivers from arm-level passengers, and the caller-sensitivity caveats of copy-number signatures. Use when finding recurrently amplified or deleted regions in a cohort, localizing driver genes, separating focal from broad events, running GISTIC2, or extracting copy-number mutational signatures.

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