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Corrects fluorophore spillover (conventional compensation) or spectral overlap (spectral unmixing) and applies variance-stabilizing transforms (logicle/biexponential, arcsinh, log) for flow and mass cytometry. Covers spillover-matrix estimation from single-stain controls, AutoSpill, the spillover spreading matrix and why panel design (not compensation) bounds resolution, compensate-then-transform ordering, and arcsinh cofactor choice (5 for CyTOF, ~150 for fluorescence, per-channel via flowVS). Use when correcting spectral overlap, preparing data for gating/clustering, choosing logicle vs arcsinh, deciding a cofactor, or distinguishing compensation from spectral unmixing.

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Maps between gene identifier systems (Ensembl, Entrez, HGNC symbol, UniProt, RefSeq, MANE) using AnnotationDbi, biomaRt, mygene, pyensembl, and Ensembl REST. Encodes Ensembl version stripping with GENCODE _PAR_Y preservation, the Ziemann 2016 Excel autocorrect debacle and Bruford 2020 HGNC renames (SEPT*->SEPTIN*, MARCH*->MARCHF*, MARC*->MTARC*, DEC1->DELEC1), OCT4/POU5F1 alias resolution, biomaRt archive endpoints for release pinning, the `filters` (plural) gotcha, MANE Select for clinical reporting, cross-species orthology via Ensembl Compara / OMA / OrthoDB, and tx2gene construction for tximport. Use when converting gene IDs across systems, handling renamed symbols, building tx2gene, pinning to a specific Ensembl release for reproducibility, or mapping cross-species orthologs.

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Delimits putative species boundaries from molecular data within the de Queiroz 2007 unified-lineage framework using ASAP (Puillandre 2021 successor to ABGD), mPTP C++ (Kapli 2017 successor to bPTP; bPTP is Python NOT R), GMYC single/multi-threshold (Pons 2006; Fujisawa 2013), multilocus BPP v4 with prior calibration from data (NOT defaults; Yang 2015), SNAPP + BFD* for SNP delimitation, DELINEATE (Sukumaran 2021) speciation-process modeling to address Sukumaran & Knowles 2017 PNAS critique that MSC delimits structure not species, integrative-taxonomy congruence (Padial 2010; Carstens 2013), Dsuite for introgression testing before sister claims (Malinsky 2021), and Meyer & Paulay 2005 barcoding-gap-absence caveat. Use when delineating species from DNA barcoding data, resolving cryptic complexes, choosing among ASAP/mPTP/BPP/DELINEATE, calibrating BPP priors, distinguishing introgression from ILS, or applying the Sukumaran-Knowles oversplitting correction.

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Extracts, filters, annotates, and exports differential expression results from DESeq2 or edgeR with proper handling of padj=NA (independent filtering, Cook's outliers, all-zero), multiple-testing correction choice (BH vs Storey q-value vs IHW vs lfsr), TREAT vs post-hoc fold-change filtering, p-value histogram diagnostics, gene annotation via org.db/biomaRt/mygene, GSEA preranked input, ORA background construction, replication reality (Schurch 2016 small-n result), and SABV/sex-stratified reporting. Use when extracting and interpreting DE results, troubleshooting padj=NA, choosing FDR method, preparing ranked lists for pathway analysis, annotating gene IDs, or comparing DESeq2 vs edgeR outputs.

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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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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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Build volcano and MA plots from differential-expression / association results with LFC shrinkage, FDR-adjusted thresholds, sensible label placement, and axis-truncation conventions. Covers EnhancedVolcano, ggplot2, matplotlib, and the apeglm/ashr/normal shrinkage decision. Use when visualizing differential-expression results (RNA-seq, ChIP-seq, ATAC-seq, proteomics) or any per-feature effect-size + p-value table.

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Search NCBI databases using Biopython Bio.Entrez (ESearch, EInfo, EGQuery, ESpell). Use when finding records by keyword, building reproducible field-qualified queries, navigating the Entrez Query Translator, exploiting the history server for large result sets, handling retmax caps, or interpreting weekly index lag. Covers PubMed, Nucleotide, Protein, Gene, SRA, GEO, Assembly, Taxonomy, ClinVar, dbSNP.

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Visualize biological networks (PPI, gene-regulatory, co-expression, pathway) with layout algorithm choice (ForceAtlas2, Fruchterman-Reingold, Kamada-Kawai, hive plots), edge bundling, community-based coloring, and reproducible seeds using NetworkX, PyVis, igraph, and Cytoscape automation. Use when rendering biological networks for static publication, interactive HTML exploration, or Cytoscape-format export.

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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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Build interactive HTML/web visualizations with plotly (Python/R), bokeh (Python), and gganimate/plotly frames for animation, with awareness of current Kaleido static-export model (post-orca-EOL), HTML file-size bloat, and the limits of interactive-only output for journal submission. Use when producing zoomable/hoverable plots for notebook EDA, supplementary HTML, dashboards, or animated time-course / iteration visualizations.

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Download genome assemblies, gene records, and ortholog data from NCBI using the modern Datasets v2 CLI (replaces assembly_summary.txt scraping and many EFetch workflows). Use when bulk-pulling genome assemblies, gene metadata across species, ortholog sets, or BLAST databases; when E-utilities are too slow for genome-scale work; or when automatic checksum verification, parallel download, and clean accession-driven retrieval are required. Encodes the JSON-lines output format, dataformat conversion, --dehydrated for cloud workflows, and when Datasets is/isn't the right tool.

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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 protein-protein and gene interaction databases (STRING, BioGRID, IntAct, SIGNOR, Reactome, HuRI, HuMAP, OmniPath, ConsensusPathDB, DIP). Use when building PPI networks, choosing between physical vs functional vs genetic interactions, signed/directed vs undirected, high-throughput vs curated, picking confidence thresholds, aggregating across resources, or navigating license constraints. Encodes the database decision matrix, STRING v12 channel semantics, OmniPath as meta-database, SIGNOR for signed signaling, and per-resource rate limits.

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Add p-value brackets, significance asterisks, and effect-size annotations to distribution plots using ggpubr, ggsignif, and statannotations with correct test selection (parametric vs non-parametric vs paired), multiple-testing adjustment, and rendering of negative results. Use when a boxplot/violin/raincloud needs in-figure statistical comparisons between groups.

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Identifies essential genes from CRISPR-Cas9 fitness screens using BAGEL2 (Kim & Hart 2021 Genome Med), a Bayesian classifier scoring per-gene Bayes Factors via log-likelihood ratios over per-sgRNA fold changes, calibrated against CEGv2 core-essentials (Hart 2017 G3, ~684 genes) and NEGv1 non-essentials (Hart 2014, ~927 genes). Covers the fc + bf + pr workflow, the linear-extrapolation improvement over BAGEL1 truncation, multi-target off-target correction, tumor-suppressor sensitivity (BAGEL2 detects enrichment), and BF-to-FDR calibration (BF >6 ≈ FDR 0.05 from Hart 2017). Use when classifying essential vs non-essential genes, calibrating BAGEL2 thresholds against PR curves, identifying tumor suppressors alongside essentials, comparing BAGEL2 hits to MAGeCK / drugZ, or generating publication-quality essentiality calls.

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Designs and analyzes pooled prime-editor (PE) screens for installing precise genetic variants without bystander confounding. Covers pegRNA design with PRIDICT and PRIDICT2 (Mathis 2023/2024) for predicting per-pegRNA editing efficiency, pegRNA architecture (spacer + scaffold + PBS + RTT), PE2 / PE3 / PE3b / PEmax / PEAR variants, MOSAIC in situ saturation mutagenesis (Hsu JY et al 2024 bioRxiv), the PRIME pooled-screen methodology (Erwood/Doman 2023 Nat Biotechnol 41:885; ~3,699 ClinVar variant screens), chromatin context as a primary determinant of PE efficiency, scaffold-incorporation and indel byproduct quantification with CRISPResso2, and the cross-modal validation strategy of PE + base-editor screens for variant function. Use when designing a pegRNA library for variant installation, choosing between BE and PE for a specific edit, predicting pegRNA efficiency before library synthesis, analyzing PE screen output, distinguishing intended-edit from scaffold-incorporation, or scaling PE screens to thousands of variants.

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Batch effect correction for CRISPR screens covering ComBat empirical-Bayes, RUV, SVA, control-sgRNA normalization, and the model-based alternative of including batch as a covariate in MAGeCK MLE or Chronos. Covers screen-specific batch sources (passage cohort, library lot, infection day, sequencing run, Cas9 lot, FBS lot), PCA + variance-decomposition diagnostic to decide if correction is needed, when correction harms biology by over-correcting condition into batch, limma removeBatchEffect for visualization-only correction, and relationship to multi-condition design matrices. Use when combining screens for joint analysis, when passage cohort confounds biology, when DepMap-style panels need Chronos with batch covariates, when picking ComBat vs RUV, or when correction harms biology and should be replaced with explicit covariate modeling.

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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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Detect syntenic blocks and structural rearrangements between genomes using MCScanX (Wang 2012), JCVI/MCScan (Tang 2008 Python), GENESPACE (Lovell 2022) for orthology-anchored riparian visualization, SyRI for structural variation, AnchorWave for sequence-level synteny, i-ADHoRe 3.0 for highly diverged species, SynNet for synteny networks, and ntSynt for multi-genome macrosynteny. Use when identifying collinear gene blocks across species, distinguishing macrosynteny from microsynteny, detecting inversions/translocations/duplications, anchoring orthology in WGD lineages, producing publication riparian plots, computing synteny block age via Ks (cross-references whole-genome-duplication), or running synteny-aware ortholog inference in polyploids.

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Infer integer allele-specific copy number, tumor purity, and ploidy from tumor sequencing by jointly modeling read depth (logR) and B-allele frequency (BAF) with ASCAT, Sequenza, FACETS, PURPLE, and PureCN (tumor-only). Covers the purity-ploidy identifiability problem, the diploid-baseline (dipLogR) anchor, major/minor copy number, loss of heterozygosity, sunrise/contour fit diagnostics, and reconciliation of conflicting fits. Use when tumor analysis needs absolute copy number rather than relative log2, when estimating purity and ploidy, calling LOH or copy-neutral LOH, resolving whole-genome doubling, running tumor-only allele-specific calling, or choosing among ASCAT, Sequenza, FACETS, and PureCN.

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Classify constitutional (germline) copy number variants for clinical reporting using the 2019 ACMG/ClinGen technical standards points-based framework, with ClassifyCNV and AnnotSV for semi-automated scoring. Covers the separate copy-number-loss and copy-number-gain rubrics, the five-tier classification, ClinGen haploinsufficiency/triplosensitivity and dosage-sensitive regions, de novo and segregation evidence, and population-frequency benign evidence. Use when assigning pathogenic/likely-pathogenic/VUS/likely-benign/benign to a constitutional CNV, scoring a CNV against ACMG/ClinGen criteria, or distinguishing the automatable evidence from the case-specific evidence requiring manual input.

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Quantify homologous recombination deficiency (HRD) from tumor copy number using the three genomic-scar metrics — loss of heterozygosity (LOH), large-scale state transitions (LST), and telomeric allelic imbalance (TAI) — with scarHRD, and via the whole-genome HRDetect and CHORD models. Covers the genomic instability score, the PARP-inhibitor clinical context, whole-genome-doubling correction, and the scar-versus-state distinction. Use when computing an HRD score for PARP-inhibitor eligibility, deriving LOH/LST/TAI scars from allele-specific copy number, deciding between scar-based and mutational-signature HRD methods, or interpreting an HRD result in a BRCA-reverted or low-purity tumor.

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Detect single-nucleotide crosslink (CL) sites in CLIP-seq data using truncation patterns (iCLIP/eCLIP CITS), crosslink-induced mutations (HITS-CLIP CIMS deletions, PAR-CLIP T-to-C), or HMM/kernel-density methods (PureCLIP, PARalyzer, CTK). Use when single-nucleotide resolution is required for motif registration (mCross), allele-specific binding (BEAPR), variant-effect prediction, or comparing crosslink chemistry across CLIP variants.

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