Identify tumor neoantigens from somatic variants with pVACtools (pVACseq/pVACfuse/pVACbind/pVACvector/pVACview) for personalized cancer vaccines and checkpoint biomarkers. Encodes the field's hard truth that binding prediction is the easy, near-solved part and single-digit-percent PPV lives downstream — so it centers clonality/CCF, HLA LOH (the silent invalidator), expression, proximal-variant phasing, agretopicity/foreignness quality, and the predicted->presented->immunogenic validation tiers. Use when nominating vaccine targets, ranking neoantigens, or building a tumor-to-candidate pipeline. Binding details in mhc-binding-prediction; ranking in immunogenicity-scoring.

Aligns Oxford Nanopore and PacBio long reads (and assemblies) to a reference with minimap2 using the error-rate-matched preset (map-ont, lr:hq, map-hifi, map-pb, splice/splice:hq, asm5/10/20, ava), producing a sorted/indexed BAM for variant, SV, methylation, or isoform analysis. Covers why the preset rewrites the scoring/chaining model, why SV calling rides on supplementary not secondary alignments, carrying MM/ML methylation tags through with -y, the multi-part-index MAPQ trap, and when to swap in Winnowmap/VACmap/lra/pbmm2. Use when mapping ONT or PacBio reads, choosing a minimap2 preset by platform/chemistry, preparing input for Clair3/medaka/Sniffles/modkit, aligning into repeats/centromeres, or spliced-aligning cDNA/Iso-Seq.

Run Bayesian phylogenetic analysis with MrBayes, BEAST2, RevBayes, and PhyloBayes including MCMC convergence diagnostics and model comparison. Use when needing posterior probability support, Bayesian model averaging, site-heterogeneous models for deep phylogenies, or formal model comparison via stepping-stone sampling.

Models continuous temporal trajectories from bulk or time-resolved omics data using generalized additive models (mgcv), spline regression, and changepoint detection (segmented, ruptures). Fits smooth gene expression curves and tests trajectory differences between conditions. Use when fitting non-linear temporal models to bulk time-series data or comparing developmental trajectories across conditions. Not for single-cell pseudotime (see single-cell/trajectory-inference).

Comprehensive variant annotation using bcftools annotate/csq, VEP, SnpEff, and ANNOVAR. Add database annotations, predict functional consequences, and assess clinical significance with MANE transcript selection and pathogenicity scoring. Use when annotating variants with functional and clinical information.

Performs structure-based virtual screening using AutoDock Vina, SMINA, GNINA (CNN scoring), and DiffDock-L hybrid workflows with explicit choice rules across rigid vs flexible docking, cross-docking vs self-docking, binding-site detection (P2Rank, fpocket), receptor preparation (PDB2PQR, PROPKA), ligand preparation (meeko, OpenBabel), and ultralarge-library screening (ZINC22, Enamine REAL). Use when screening chemical libraries against a protein target to find candidate binders, ranking docking poses, or selecting a docking workflow for a specific scenario.

End-to-end ATAC-seq workflow from FASTQ files to differential accessibility and TF footprinting. Covers alignment, peak calling with MACS3, QC metrics, and optional TOBIAS footprinting. Use when running end-to-end ATAC-seq analysis from FASTQ to differential accessibility.

End-to-end ChIP-seq workflow from FASTQ files to annotated peaks. Covers QC, alignment, peak calling with MACS3 (or HOMER), and peak annotation with ChIPseeker. Use when processing ChIP-seq data from alignment through peak annotation.

Profiles the functional potential of shotgun metagenomes with HUMAnN 3's tiered search (MetaPhlAn prescreen, Bowtie2 pangenome, translated DIAMOND vs UniRef), giving gene-family (RPK) and MetaCyc pathway abundances stratified by species. Covers why a metagenome measures potential not activity, why dropping UNMAPPED/UNINTEGRATED biases everything, why stratification is an estimate, coverage-vs-abundance and MinPath/gap-fill, UniRef90-vs-50 and biome database bias, and the assembly/eggNOG/dbCAN/antiSMASH alternatives. Use when obtaining pathway or gene-family abundances, regrouping to KO/EC/GO, normalizing functional tables, or choosing read-based vs assembly-based functional profiling. For AMR genes see amr-detection; for host-gene enrichment see pathway-analysis.

End-to-end genome annotation pipeline from assembled contigs to functional annotation, covering repeat masking, gene prediction, and functional assignment for both prokaryotic and eukaryotic genomes. Use when annotating a newly assembled genome from scratch.

Generates 3D conformer ensembles using RDKit ETKDGv3 with knowledge-enhanced distance geometry, MMFF94/UFF force-field optimization, CREST + GFN2-xTB semi-empirical refinement, and macrocycle-aware torsion preferences. Provides explicit decision rules for single vs ensemble conformer use, RMSD pruning, energy windows, conformer count, and force-field choice. Use when preparing 3D ligands for docking, generating descriptor input for 3D QSAR, or sampling macrocycle/peptide conformational ensembles.

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.

Build UpSet plots to visualize set intersections beyond 4 sets (where Venn fails) using ComplexUpset (modern, ggplot2-grammar) or the unmaintained UpSetR, with explicit cardinality vs degree sorting, attribute panels, and query highlighting. Use when comparing overlap across many gene sets, peak sets, variant lists, or any set membership matrix where Venn diagrams become illegible.

Infer gene regulatory networks from bulk or general expression data with mutual-information (ARACNe) and tree-ensemble (GENIE3, GRNBoost2) methods, and infer transcription-factor protein activity from regulons with VIPER and msVIPER. Covers the activity-not-edges paradigm, the undirected-association caveat, the DREAM5 wisdom-of-crowds and method-complementarity result, AUPRC-over-AUROC evaluation, and gold-standard incompleteness. Use when inferring a regulatory network from a bulk expression matrix, finding master regulators, or scoring TF activity from a signature. For single-cell motif-pruned regulons see scenic-regulons; for co-expression modules see coexpression-networks.

Load and preprocess imaging mass cytometry (IMC) and MIBI data from raw MCD/TXT through hot-pixel removal, spillover compensation, and variance-stabilizing transformation, covering readimc/steinbock ingestion, NNLS spillover compensation (CATALYST), IMC-Denoise, and the IMC arcsinh-cofactor question. Use when starting analysis from raw MCD files, building per-channel TIFF stacks, compensating channel spillover, choosing an arcsinh cofactor, or preparing single-cell intensities for phenotyping.

Rank and prioritize neoantigen/epitope candidates by likely T-cell response using NeoFox feature annotation, PRIME2.0, BigMHC-IM, the Łuksza/Balachandran fitness model (agretopicity + foreignness), and pVACtools tiering. Encodes the field's hard truths that immunogenicity is the least-solved layer (dedicated scores ~AUROC 0.6-0.7, modest PPV), that scores are valid only for RANKING within one patient (never absolute go/no-go or cross-patient), that DAI has anchor-inflation and WT-denominator traps, and that stacking weak correlated scores into one number is a red flag. Use when ordering a candidate list for a vaccine. Binding lives in mhc-binding-prediction; calling in neoantigen-prediction.

Polishes Oxford Nanopore draft assemblies to higher consensus accuracy with medaka, a basecaller-model-specific neural consensus net, produces haploid variant calls (VCF) for microbial, mitochondrial, or viral samples, and generates amplicon/viral consensus sequences. Covers the model-matching footgun that silently degrades output, why Racon-first is obsolete and medaka runs directly on Flye output as a single pass, why HiFi must never be fed to medaka, the v1->v2 subcommand renames, and the precise medaka_variant deprecation. Use when polishing an ONT-only assembly, generating an amplicon/viral consensus, calling a haploid ONT consensus, or deciding whether medaka, dorado polish, or Clair3 is the right tool.

Detects structural variants (deletions, insertions, inversions, duplications, translocations) from Oxford Nanopore and PacBio long-read alignments with Sniffles2, cuteSV, SVIM, and assembly-based callers, joint-genotypes cohorts via the Sniffles2 .snf workflow, and benchmarks with Truvari against GIAB. Covers why an SV call is a representation artifact (the tandem-repeat BED, aligner, and Truvari params set precision/recall as much as the caller), the cuteSV per-platform parameter trap, soft-clipped supplementary alignments as the SV substrate, and the somatic/mosaic boundary to Severus/nanomonsv. Use when calling germline or somatic SVs from ONT/HiFi reads, joint-genotyping a cohort, choosing or tuning an SV caller, or benchmarking SV calls.

Builds and applies 3D pharmacophore models using RDKit Pharm3D, the apo2ph4 receptor-based workflow (Heider et al 2022/2023 J Chem Inf Model 63:147-158), Pharmer / Pharmit (search), and PharmacoForge (diffusion-based generation, Flynn et al 2025 Front Bioinform), covering ligand-based pharmacophore (from active set alignment) and receptor-based pharmacophore (from binding pocket geometry). Explicit handling of feature types, geometric tolerances, partial matching, and pharmacophore-based virtual screening. Use when identifying scaffold-hopping candidates, building shape-and-feature search queries, or transferring SAR across chemotypes.

Statistical testing for differentially abundant proteins between conditions. Covers preprocessing (log2 transformation, normalization), limma and DEqMS workflows with empirical Bayes moderation, fold change shrinkage for accurate effect size estimation, and Python alternatives. Use when identifying proteins with significant abundance changes between experimental groups.

Builds QSAR / QSPR models using chemprop D-MPNN, MolFormer, Uni-Mol, ChemBERTa, random forest baselines, and Gaussian processes with explicit handling of OECD 5 principles, applicability domain (kNN, leverage, conformal prediction, Mahalanobis), scaffold-balanced splits, ensemble uncertainty, calibration (Platt, isotonic), feature importance (SHAP, atomic attribution), and prospective validation. Use when building target-specific predictive models from in-house bioassay data, ADMET endpoints, or selectivity profiles.

Analyzes experimental RNA structure probing data from SHAPE-MaP and DMS-MaPseq experiments using ShapeMapper2. Converts mutation rates to per-nucleotide reactivity profiles that constrain structure prediction. Use when processing SHAPE-MaP or DMS-MaPseq sequencing data to obtain experimental RNA structure information.

Predicts ADMET properties using ADMETlab 3.0 (119 endpoints with uncertainty), ADMET-AI, DeepChem MolNet, and chemprop D-MPNN with explicit handling of OECD QSAR principles, applicability domain assessment, calibration, hERG/CYP/AMES gold-standard endpoints, and PAINS / Lipinski / Ro5 / Veber / BBB druglikeness filters. Use when filtering compounds for drug-likeness, prioritizing leads by predicted safety, or building an in-house ADMET QSAR model.

Build OncoPrint and co-mutation matrix plots from somatic-variant cohorts using ComplexHeatmap, maftools, and comut.py with alteration-type stacking, sample ordering by mutational burden, mutual-exclusivity overlays, and clinical annotation tracks. Use when visualizing per-sample mutation patterns across recurrent driver genes, comparing alteration classes, or identifying mutually-exclusive / co-occurring driver pairs.

Variant calling with GATK HaplotypeCaller following best practices. Covers germline SNP/indel calling, GVCF workflow for cohorts, joint genotyping, and variant quality score recalibration (VQSR). Use when calling variants with GATK HaplotypeCaller.

Predict peptide-MHC class I binding and natural presentation with MHCflurry, NetMHCpan-4.1, and MixMHCpred to nominate candidate CD8 T-cell epitopes. Covers the binding-affinity (BA) vs eluted-ligand (EL/presentation) distinction, why %Rank beats raw nM for cross-allele work, the MS abundance bias that misranks low-expression neoantigens, allele-coverage inequity, and length bias. Use when scanning a protein or peptide set for class I epitopes, scoring neoantigen candidates, or choosing a binding predictor. For CD4/HLA class II see mhc-class-ii-prediction.

Calls DNA base modifications (5mC, 5hmC, 6mA, 4mC) directly from Oxford Nanopore and PacBio HiFi long reads encoded as MM/ML SAM tags, piles them into per-site bedMethyl with modkit (or pb-CpG-tools for PacBio), and produces phased allele-specific methylation. Covers why methylation is a basecalling decision that cannot be recovered later, the MM/ML tag-drop failure that silently zeroes methylation through alignment, the MM ? vs . no-call semantics, 5mC/5hmC resolution vs bisulfite, modkit's 10th-percentile auto-threshold, and the haplotagged ASM workflow. Use when calling 5mC/5hmC/6mA from a modBAM, generating bedMethyl, preserving methylation tags through alignment, doing allele-specific or differential methylation, or QC-ing a modification BAM.

Standardizes molecular structures using ChEMBL chembl_structure_pipeline and RDKit rdMolStandardize covering sanitization, salt/solvent stripping, neutralization, tautomer canonicalization, stereochemistry standardization, mixture handling, and isotope normalization. Explicitly compares ChEMBL pipeline, canSARchem, and PubChem standardization choices. Use when preparing libraries for QSAR training, joining datasets across sources, deduplicating compound collections, or building canonical compound registries.

Estimate divergence times using molecular clock models with BEAST2, MCMCTree, and TreePL. Use when dating speciation events, calibrating phylogenies with fossils, choosing between strict and relaxed clock models, or estimating evolutionary rates across lineages.

Enumerates virtual chemical libraries via reaction SMARTS transformations using RDKit and Reaction templates, with explicit handling of atom mapping, template extraction (RDKit reaction mining), product validation, RECAP/BRICS fragmentation, R-group decomposition, matched molecular pair analysis (MMPA), and Free-Wilson analysis. Use when generating combinatorial libraries from building blocks, enumerating analog series, deriving structure-activity rules, or extracting transformations from reaction data.

Detects circadian and ultradian rhythms in time-series omics data using CosinorPy cosinor models, MetaCycle (JTK_CYCLE, ARSER), and RAIN non-parametric tests. Fits cosine models to estimate phase and amplitude, tests rhythmicity significance at pre-specified periods. Use when testing for 24-hour or other known-period oscillations in circadian, feeding-fasting, or light-dark cycle experiments. Not for unknown-period discovery (see temporal-genomics/periodicity-detection).

Generate variant statistics, sample concordance, and quality metrics using bcftools stats and gtcheck. Use when evaluating variant quality, comparing samples, or summarizing VCF contents.

End-to-end DNA sequencing workflow from FASTQ files to variant calls. Covers QC, alignment with BWA, BAM processing, and variant calling with bcftools or GATK HaplotypeCaller. Use when calling variants from raw sequencing reads.

Quality control for flow, spectral, and mass cytometry - time-based anomaly cleaning (flowAI, flowCut, PeacoQC, flowClean), margin/boundary event removal, signal-drift detection, dead-cell exclusion, CyTOF Gaussian/DNA/event-length checks, instrument calibration/standardization (MESF, CS&T, peak-2), and batch-level outlier flagging. Use when assessing acquisition quality, choosing a cleaning tool, ordering QC relative to compensation, deciding margin removal before density-based steps, or flagging problematic samples before clustering or differential analysis.

Segment single cells from multiplexed IMC/MIBI tissue images using Mesmer/DeepCell, Cellpose, or ilastik+CellProfiler, covering whole-cell vs nuclear segmentation, the summed-membrane-channel decision, nuclear-expansion bias, lateral spillover, resolution-floor parameters, and downstream-proxy evaluation. Use when delineating cells after preprocessing, choosing a segmentation model, building a cell mask for quantification, diagnosing impossible double-positive populations, or troubleshooting over/under-segmentation.

Analyze spatial cell-cell interactions, neighborhoods, and niches in IMC/MIBI data with squidpy and imcRtools, covering neighborhood-enrichment permutation nulls, the abundance-vs-density confound, inhomogeneous Ripley's K, cellular-neighborhood discovery, graph-construction (contact vs proximity), and edge effects. Use when testing whether cell types co-locate, choosing a spatial null, building a neighbor graph, discovering tissue niches, or deciding whether a spatial pattern is real or a density/segmentation artifact.

Predict peptide-MHC class II (HLA-DR/DQ/DP) binding and presentation for CD4 T-cell epitopes with NetMHCIIpan-4.3 and MixMHC2pred-2.0. Covers why class II is far less reliable than class I (open binding groove, 9-mer register ambiguity, sparse noisy training data, DR>DP>DQ accuracy asymmetry), the DQ/DP heterodimer alpha/beta pairing trap, and the looser 1%/5% %Rank thresholds. Use when predicting CD4 epitopes for vaccine help, mapping class II neoantigens, or scoring long peptides against DR/DQ/DP. For CD8/class I see mhc-binding-prediction.

Calls germline small variants (SNPs and indels) from Oxford Nanopore and PacBio HiFi long reads with Clair3, a two-stage (pileup + full-alignment) deep-learning caller, selecting the chemistry- and basecaller-version-matched model, enabling read-based phasing, and benchmarking against GIAB with stratification. Covers why the model string is the experiment (no auto-detection, silent degradation on mismatch), why ONT homopolymer/STR indels are the residual error whole-genome F1 hides, and the somatic/trio/RNA boundary to the ClairS/Clair3-Trio family. Use when calling germline SNVs/indels from ONT or HiFi BAMs, choosing a Clair3 model, phasing variants, or benchmarking long-read calls.

Build maximum likelihood phylogenetic trees using IQ-TREE2 and RAxML-NG with expert model selection, branch support assessment, and topology testing. Use when inferring publication-quality ML trees, selecting substitution models, interpreting bootstrap and concordance factor support, or running partitioned phylogenomic analyses.

Generate pileup data for variant calling using samtools mpileup and pysam. Use when preparing data for variant calling, analyzing per-position read data, or calculating allele frequencies.

Clusters genes by temporal expression profile shape using Mfuzz soft clustering, TCseq, and DEGreport degPatterns. Groups co-regulated genes into shared trajectory patterns via fuzzy c-means or hierarchical approaches. Use when categorizing temporally dynamic genes into response groups or identifying co-expression modules across time points. Requires temporally variable genes identified first (see differential-expression/timeseries-de).

Clinical variant interpretation using ClinVar, ACMG guidelines, and pathogenicity predictors. Prioritize variants for diagnostic and research applications. Use when interpreting clinical significance of variants.

Merge, concatenate, sort, intersect, and subset VCF files using bcftools. Use when combining variant files, comparing call sets, or restructuring VCF data.

End-to-end biomarker discovery workflow from expression data to validated biomarker panels. Covers feature selection with Boruta/LASSO, leakage-safe cross-validation, calibration, and SHAP interpretation. Use when building and validating diagnostic or prognostic biomarker signatures from omics data.

End-to-end single-cell RNA-seq workflow from 10X Genomics data to annotated cell types. Covers QC, normalization, clustering, marker detection, and cell type annotation. Use when analyzing single-cell RNA-seq data.

Filter alignments by flags, mapping quality, and regions using samtools view and pysam. Use when extracting specific reads, removing low-quality alignments, or subsetting to target regions.

Generate alignment statistics using samtools flagstat, stats, depth, coverage, and mosdepth. Use when assessing alignment quality, calculating coverage, or generating QC reports.

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.

Plot per-gene mutation distributions on a protein-domain map (lollipop / needle plots) showing mutation position, recurrence count, and variant classification with maftools, g3-lollipop, trackViewer, and ProteinPaint. Use when visualizing recurrent mutation hotspots on a single gene's protein, marking domain boundaries from UniProt/Pfam, comparing missense vs truncating distributions, or contrasting two cohorts on the same lollipop.

Assesses Oxford Nanopore and PacBio long-read quality with NanoPlot, cramino, NanoComp, pycoQC/toulligQC, and seqkit, and filters reads with chopper/Filtlong for the downstream goal. Covers why read-only Qscore is an uncalibrated posterior (real accuracy needs a reference BAM), why the sequencing_summary.txt is required for run-health metrics, intent-conditioned filtering (preserve long reads and small replicons for assembly, filter almost nothing for variant calling), the chimera/internal-adapter trap that fabricates SVs, and PacBio rq-based HiFi QC. Use when judging a long-read run, computing read N50 or percent identity, filtering reads before assembly or variant calling, comparing barcodes/runs, or reading run-health red flags.

Find patterns, motifs, and subsequences in biological sequences using Biopython. Use when searching for transcription factor binding sites, regulatory elements, or any sequence pattern. For restriction enzyme analysis, use the restriction-analysis skill.

Searches for non-coding RNA homologs and classifies RNA families using Infernal covariance model searches against the Rfam database. Identifies structured RNAs by sequence and secondary structure conservation. Use when querying sequences against Rfam, building custom covariance models for novel RNA families, or classifying non-coding transcripts by family.

Predicts RNA secondary structures using minimum free energy folding and partition function analysis with ViennaRNA (RNAfold, RNAalifold, RNAcofold). Computes base-pair probabilities, centroid structures, and consensus structures from alignments. Use when predicting RNA folding, evaluating structural stability, or comparing structures across homologs.

Normalize indel representation, decompose MNPs, and split multiallelic variants using bcftools norm. Use when comparing variants from different callers, preparing VCF for database annotation, or merging VCFs from multiple sources.

End-to-end Hi-C analysis workflow from FASTQ to compartments, TADs, and loops, with the decision of WHICH features the sequencing depth can support. Covers pairtools read-pair processing and library QC, cooler matrices, ICE balancing and distance-decay expected, A/B compartments, TAD boundaries, loop calling, and the routing of HiChIP/PLAC-seq/Capture Hi-C to protein-directed loop callers. Use when processing Hi-C data end to end, deciding a resolution for a given depth, or choosing between bulk-Hi-C and protein-directed loop calling.

Analyze codon usage, calculate CAI (Codon Adaptation Index), and examine synonymous codon bias using Biopython. Use when analyzing coding sequences for expression optimization or evolutionary analysis.

Build Sankey, alluvial, river, and CONSORT-style flow diagrams to visualize cohort transitions, cell-state changes, or pipeline filtering using ggalluvial, networkD3, plotly, and consort. Use when showing how entities move between categories across timepoints (cell states, drug response classes, patient flow through a trial) or filtering pipelines (variants filtered through QC stages).

Build genome-browser-style multi-track figures with pyGenomeTracks (config-driven), Gviz (R), and IGV batch screenshotting. Covers BigWig coverage tracks, BED/peak overlays, gene-model rendering, Hi-C matrix tracks, BedPE link arcs, spike-in-aware normalization, and the bamCoverage --normalizeUsing trap. Use when producing publication figures of genomic loci with stacked aligned tracks (coverage, peaks, genes, interactions) for ChIP-seq, ATAC-seq, RNA-seq, Hi-C, or generic locus visualization.

Build sequence logos from aligned DNA, RNA, or protein motifs using ggseqlogo (R), Logomaker (Python), or WebLogo with explicit bits vs probability encoding, background-frequency correction, custom alphabets, and multi-logo stacking. Use when visualizing motif PWMs (TF binding, splice sites, CRISPR spacers), aligned-position composition, or comparing two motif sets.

Reads, inspects, and writes Flow Cytometry Standard (FCS) files from conventional, spectral, and mass cytometry (CyTOF), and parses FlowJo/Cytobank/Diva workspaces. Covers FCS 2.0/3.0/3.1/3.2 internals ($PnE linear-vs-log, $DATATYPE, $SPILLOVER vs SPILL vs $COMP, $TIMESTEP), channel/parameter metadata, the silent linearize/truncate defaults, and R (flowCore, flowWorkspace, CytoML) plus Python (FlowKit, readfcs) readers. Use when loading flow or mass cytometry data, mapping detector channels to antibodies, extracting the event matrix, choosing a reader, or bridging FCS to the scanpy/AnnData ecosystem before preprocessing.

Reads, writes, and converts molecular file formats (SMILES, InChI, SDF V2000/V3000, MOL2, PDB, MMTF) using RDKit and Open Babel with rigorous handling of aromaticity perception, stereochemistry, implicit/explicit hydrogens, kekulization, and salt/fragment separation. Use when loading chemical libraries, debugging parse failures, or preparing molecules for downstream standardization, descriptor calculation, or docking.

Validates docked / generated protein-ligand poses using PoseBusters physical-validity tests, strain energy quantification, geometric checks (planarity, vdW overlap, bond/angle distortion), and pose-energy reasonableness. Filters AI-docking outputs (DiffDock, EquiBind, NeuralPLexer) where ~50% of poses fail physical-validity tests. Use when QC-ing docking results, comparing classical vs ML docking outputs, or filtering pose lists before SAR analysis.

View, convert, and understand SAM/BAM/CRAM alignment files using samtools and pysam. Use when inspecting alignments, converting between formats, or understanding alignment file structure.

Performs 3D shape-based similarity searching using ROCS (OpenEye), USRCAT (ultra-fast), Open3DAlign (RDKit), ESPSim (electrostatic), and ShaEP with explicit handling of Tanimoto-Combo (shape + color), shape vs ECFP4 complementarity, conformer-ensemble searching, alignment optimization, and scaffold hopping. Use when searching for shape-mimicking compounds with different scaffolds, identifying bioisosteric replacements, prospective scaffold hopping, or expanding hit series beyond 2D similarity.

End-to-end alternative splicing analysis from FASTQ to differential splicing results for short-read bulk RNA-seq. Aligns with STAR 2-pass cohort-style, performs junction QC (RSeQC, MaxEntScan, SpliceAI), runs rMATS-turbo and leafcutter for concordant differential analysis, optionally MAJIQ V3 for complex events / heterogeneous cohorts, isoform-switching with NMD/ORF/domain consequences (IsoformSwitchAnalyzeR v2 + DRIMSeq+DEXSeq+stageR DTU), and sashimi visualizations. Use when performing comprehensive splicing analysis from raw bulk RNA-seq data; for variant-driven splice prediction see splice-variant-prediction; for rare-disease single-patient outlier detection see outlier-splicing-detection; for full-isoform PacBio/ONT analysis see long-read-splicing.

Call structural variants (SVs) from sequencing data using Manta, Delly, GRIDSS, and LUMPY. Detects deletions, insertions, inversions, duplications, and translocations too large for standard SNV callers. Use when detecting structural variants from short-read or long-read data and building consensus callsets.

Workflow from differential expression results to functional enrichment analysis. Covers GO, KEGG, Reactome enrichment with clusterProfiler and visualization. Use when taking DE results to pathway enrichment.

End-to-end multi-omics integration workflow. Orchestrates data harmonization, MOFA/mixOmics integration, factor interpretation, and downstream analysis across transcriptomics, proteomics, metabolomics, and other modalities. Use when integrating multiple omics datasets.

End-to-end RNA-seq workflow from FASTQ files to differential expression results. Covers QC, quantification (Salmon or STAR+featureCounts), and DESeq2 analysis with visualization. Use when running RNA-seq from FASTQ to DE results.

Generate consensus FASTA sequences by applying VCF variants to a reference using bcftools consensus. Use when creating sample-specific reference sequences or reconstructing haplotypes.

Estimate species trees using coalescent methods including ASTRAL-III, wASTRAL, ASTRAL-Pro, SVDQuartets, and BPP. Use when multi-locus data shows gene tree discordance from incomplete lineage sorting, when in the anomaly zone where concatenation is misleading, or when computing concordance factors to assess topological support.

Select restriction enzymes by criteria using Biopython Bio.Restriction. Find enzymes that cut once, don't cut, produce specific overhangs, are commercially available, or have compatible ends for cloning. Use when selecting restriction enzymes for cloning or analysis.

Build publication-quality figures in R with ggplot2 using the grammar of graphics (data + aesthetics + geometries + scales + facets + themes) with CVD-safe palettes, cairo_pdf TrueType embedding, programmatic aes via tidy evaluation, and the theme_classic publication baseline. Use when producing static figures in R for papers, presentations, or reports.

Mark and remove PCR/optical duplicates using samtools fixmate and markdup. Use when preparing alignments for variant calling or when duplicate reads would bias analysis.

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.

Cell-free DNA analysis pipeline from plasma sequencing to tumor monitoring. Preprocesses cfDNA reads, analyzes fragment patterns, estimates tumor fraction from sWGS, and optionally detects mutations from targeted panels. Use when analyzing liquid biopsy samples for cancer detection or monitoring.

Perform geometric calculations on protein structures using Biopython Bio.PDB. Use when measuring distances, angles, and dihedrals, superimposing structures, calculating RMSD, or computing solvent accessible surface area (SASA).

Compute evolutionary distances and build phylogenetic trees using Biopython Bio.Phylo.TreeConstruction. Use when creating distance matrices from alignments, building NJ/UPGMA trees, generating bootstrap consensus, or needing quick exploratory phylogenies before running full ML analysis.

Generate consensus sequences and manage reference files using samtools. Use when creating consensus from alignments, indexing references, or creating sequence dictionaries.

Single-cell ATAC-seq analysis with Signac (R/Seurat) and ArchR. Process 10X Genomics scATAC data, perform QC, dimensionality reduction, clustering, peak calling, and motif activity scoring with chromVAR. Use when analyzing single-cell ATAC-seq data.

Predict protein structures using modern ML models including AlphaFold3, ESMFold, Chai-1, and Boltz-1. Use when predicting structures for novel proteins, protein complexes, or when comparing predictions across multiple methods.

Joint genotype calling across multiple samples using GATK CombineGVCFs and GenotypeGVCFs. Essential for cohort studies, population genetics, and leveraging VQSR. Use when performing joint genotyping across multiple samples.

Detects and removes doublets/aggregates from flow, spectral, and mass cytometry before clustering or quantification. Covers FSC-A vs FSC-H singlet discrimination (the Area-Height non-proportionality, not a 1D area gate), FSC-W/SSC width gating, CyTOF Gaussian discrimination parameters (Center/Offset/Width/Residual/Event_length) and DNA intercalator gating, and the residual heterotypic conjugates that survive scatter gating and masquerade as double-positive populations. Use when filtering aggregates before phenotyping, choosing a doublet method for flow vs CyTOF, or diagnosing a suspicious double-positive cluster.

Gene Set Enrichment Analysis using clusterProfiler gseGO and gseKEGG. Use when analyzing ranked gene lists to find coordinated expression changes in gene sets without arbitrary significance cutoffs. Detects subtle but coordinated expression changes.

Modify phylogenetic tree structure using Biopython Bio.Phylo. Use when rooting trees with outgroups, midpoint, or MAD methods, pruning taxa, collapsing clades, ladderizing branches, or extracting subtrees. Includes rooting method decision guidance.

Call SNPs and indels from aligned reads using bcftools mpileup and call. Use when detecting variants from BAM files or generating VCF from alignments.

Gene Ontology over-representation analysis using clusterProfiler enrichGO. Use when identifying biological functions enriched in a gene list from differential expression or other analyses. Supports all three ontologies (BP, MF, CC), multiple ID types, and customizable statistical thresholds.

Modify protein structures using Biopython Bio.PDB. Use when transforming coordinates, removing atoms or residues, adding new entities, modifying B-factors and occupancies, or building structures programmatically.

Detect and remove doublets (multiple cells captured in one droplet) from single-cell RNA-seq data. Uses Scrublet (Python), DoubletFinder (R), and scDblFinder (R). Essential QC step before clustering to avoid artificial cell populations. Use when identifying and removing doublets from scRNA-seq data.

Deep learning-based variant calling with Google DeepVariant. Provides high accuracy for germline SNPs and indels from Illumina, PacBio, and ONT data. Use when calling variants with DeepVariant deep learning caller or when highest germline calling accuracy is required.

End-to-end 16S amplicon workflow from FASTQ reads to differential abundance. Orchestrates DADA2 ASV inference, taxonomy assignment, diversity analysis, and compositional testing with ALDEx2. Use when processing 16S/ITS amplicon data.

Visualize enrichment results using enrichplot package functions. Use when creating publication-quality figures from clusterProfiler results. Covers dotplot, barplot, cnetplot, emapplot, gseaplot2, ridgeplot, and treeplot.

KEGG pathway and module enrichment analysis using clusterProfiler enrichKEGG and enrichMKEGG. Use when identifying metabolic and signaling pathways over-represented in a gene list. Supports 4000+ organisms via KEGG online database.

Quality control of phasing and imputation results. Filter by INFO scores, assess accuracy, and prepare imputed data for downstream analysis. Use when filtering low-quality imputed variants or validating imputation accuracy before GWAS.

Integrate multiple scRNA-seq samples/batches using Harmony, scVI, Seurat anchors, and fastMNN. Remove technical variation while preserving biological differences. Use when integrating multiple scRNA-seq batches or datasets.

End-to-end multiome workflow for joint scRNA-seq + scATAC-seq analysis. Covers data loading, separate modality processing, and WNN integration with Seurat/Signac. Use when analyzing joint scRNA+scATAC data.