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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.

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Analyzes differential transcript usage (DTU) and isoform switches with functional consequence prediction (NMD via 50nt rule, ORF disruption, protein domain loss/gain, signal peptide changes, IDR alterations, coding-potential shifts). Tools include IsoformSwitchAnalyzeR v2 (auto-selects satuRn for >5 reps else DEXSeq), the manual DRIMSeq -> DEXSeq/satuRn -> stageR DTU pipeline, and fishpond/swish for inferential-uncertainty-aware DTE. Distinguishes DTU from DGE and DTE; integrates external annotators (CPC2, Pfam, SignalP, IUPred2A or DeepTMHMM). Use when investigating how splicing differences alter protein function or trigger NMD-mediated degradation.

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Analyzes alternative splicing from PacBio Iso-Seq (HiFi, Kinnex/MAS-Iso-seq) and Oxford Nanopore (direct cDNA, direct RNA, R10.4.1+) long-read RNA-seq with full-isoform resolution. Tools include FLAIR (correct/collapse/quantify/diffSplice for PacBio + ONT), IsoQuant (de-novo or annotation-guided isoform discovery 2024 SOTA), Bambu (annotation-aware Bayesian discovery + quantification with Novel Discovery Rate), SQANTI3/SQANTI-LR (isoform classification: FSM/ISM/NIC/NNC + artifact flags), rMATS-long (event calling on long-read isoforms), and minimap2 (-ax splice:hq for HiFi; -ax splice -k14 for ONT cDNA; add -uf only for direct RNA or stranded cDNA preps). Solves microexon detection, recursive splicing, complex multi-exon isoforms, and DTU without transcript-quantification uncertainty. Use when short-read AS limitations (anchor length, complex isoforms, microexons, recursive splicing, transcript ambiguity) demand full-isoform resolution.

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Analyzes alternative splicing at single-cell resolution. The first decision is library chemistry — 10X 3' is fundamentally limited (RT primes from poly-A, R2 falls in 3' UTR, <0.1 junction read per cell per AS event). Plate-based full-length methods (Smart-seq3, FLASH-seq, VASA-seq, STORM-seq) and single-cell long-read (MAS-Iso-seq, scISOr-Seq2) are the chemistries that give per-cell isoform structure. Tools include MARVEL (R, Smart-seq integrated), BRIE2 (Bayesian PSI with regulatory features and ELBO_gain test), scQuint (junction-cluster, plate-based; not for 10X), SpliZ (annotation-free Z-score), Psix (graph-smoothness regulated AS), and Sierra (alternative polyadenylation, often confused with AS). Use when analyzing isoform usage in scRNA-seq, identifying cell-type-specific splicing, or determining whether scRNA-seq chemistry supports splicing analysis at all.

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

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Predicts whether a DNA variant alters mRNA splicing using sequence-based deep-learning tools — SpliceAI (10kb context dilated CNN, clinical default), Pangolin (multi-tissue), MMSplice (modular per-region CNN with calibrated ΔPSI), SpliceTransformer/TrASPr (tissue-aware transformers), SpliceVault (empirical 300K-RNA lookup of likely mis-splicing outcomes), CADD-Splice (composite score). Applies the ClinGen SVI 2023 framework for ACMG/AMP variant interpretation (PVS1, PP3, BP4 evidence codes), HGVS splicing nomenclature (c.123+1G>A, c.123-3T>G, r.spl?), extended-window scoring for deep-intronic pseudoexons, tissue-specific predictions, branchpoint variant detection (BPHunter, LaBranchoR), and splice-switching ASO design. Use when interpreting splice impact of clinical variants, prioritizing VUS, identifying deep-intronic pathogenic variants, or designing ASOs.

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

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Create and use BAI/CSI indices for BAM/CRAM files using samtools and pysam. Use when enabling random access to alignment files or fetching specific genomic regions.

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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.

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Sort alignment files by coordinate or read name using samtools and pysam. Use when preparing BAM files for indexing, variant calling, or paired-end analysis.

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Trim PCR primers from aligned reads in amplicon-panel BAMs using samtools ampliconclip. Use when processing SARS-CoV-2 ARTIC, hereditary cancer panels, ctDNA hot-spot panels, or any amplicon assay where primer-derived bases would falsely confirm reference at primer footprints.

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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.

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Audit required bioinformatics command-line tools against Lmod or Environment Modules on Linux HPC clusters, resolve executable and version requirements, and reproducibly deploy missing software through a site-approved EasyBuild, Spack, Bioconda/micromamba, Apptainer, binary, or source-build workflow. Use when Codex must inspect module availability, diagnose a missing or wrong-version bioinformatics tool, install scientific software on Slurm/PBS clusters, publish or validate modulefiles, or produce an auditable and rollback-ready cluster software deployment.

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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.

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Secure the path from an inbound event (webhook, email, SMS, GitHub comment, file, cron) to an agent spawn in the Port Daddy fleet. Use when wiring trigger sources to agents, reviewing the io-wiring registry, adding a trust/permission gate, hardening output sinks (SSRF/path traversal), scoping tools by provenance, or proving the spawn path is safe against prompt-injection → tool-abuse. Encodes ADR-0093 and the red-team threat model. NOT for generic Coast Guard sandboxing (ADR-0050), the macaroon push-grant gate alone (ADR-0053), or non-fleet code.

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Master of text-based visual communication using ASCII art, Unicode box-drawing, and structured diagram notation. Creates clear, maintainable diagrams for systems, processes, hierarchies, relationships, and psychological structures. Proactively generates diagrams to enhance understanding. Activate on visualization needs, system architecture, process flows, psychological mapping, or when complex concepts would benefit from visual representation. NOT for photo editing, vector graphics, or GUI-based design tools.

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Expert in developer tool documentation as a product. Activate on: docs site, getting started guide, API reference, developer documentation, Docusaurus, Mintlify, Nextra, ReadMe, migration guide, troubleshooting page, docs information architecture, documentation testing, llms.txt. NOT for: marketing copy (use seo-content-blogging), API design itself (use api-architect), general technical writing (use technical-writer).

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Expert in cluttering -- the lesser-known fluency disorder characterized by rapid/irregular speech rate, excessive disfluencies, and frequent ADHD comorbidity. Covers differential diagnosis from stuttering, assessment protocols, therapy approaches (rate control, self-monitoring, pragmatic awareness), technology-assisted intervention, and AI tools for people who clutter. Activate on 'cluttering', 'rapid speech disorder', 'speech rate too fast', 'speech disfluency not stuttering', 'disorganized speech', 'ADHD speech problems', 'cluttering vs stuttering', 'speech intelligibility', 'mazed speech', 'telescoping words'. NOT for stuttering-only treatment (overlap is common but they are distinct), hearing disorders, or voice disorders (pitch, resonance).

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Send and receive transactional emails with Cloudflare Email Service (Email Sending + Email Routing). Use when building email sending (Workers binding or REST API), email routing, Agents SDK email handling, or integrating email into any app — Workers, Node.js, Python, Go, etc. Also use for email deliverability, SPF/DKIM/DMARC, wrangler email setup, MCP email tools, or when a coding agent needs to send emails. Even for simple requests like "add email to my Worker" — this skill has critical config details.

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Cooperative vibe coding across machines — real-time collaborative development with humans and AI agents. Covers current tools (Live Share, Code With Me, Zed, Cursor multiplayer, tmux), git-based async patterns, AI agent coordination, and the aspirational future of multi-human multi-agent development. Activate on: "pair programming", "vibe coding together", "collaborative coding", "mob programming", "remote pairing", "code together", "multiplayer coding", "Live Share", "Code With Me", "ensemble programming", "cooperative development". NOT for: building a real-time collaboration ENGINE (use real-time-collaboration-engine), git workflow mechanics (use git-best-practices), single-user AI coding assistance.

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Generates shell completion scripts for CLI tools. Parses command structure from help output, commander/yargs configs, or manual specification. Produces completions for bash, zsh, and fish that follow each shell's conventions.

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Audit whether an enforced-coordination MCP has actually shrunk its ~19 legacy etiquette verbs into the 5 enforced tools — work/act/ask/recall/status — each carrying one denial shape and one transcript event, migrated through a real retirement path (intake-metadata, alias, or doc-history — never a parallel runtime), with zero legacy-verb calls once a body reaches compliance mode C4+. Use when reviewing a broker-collapse migration plan, gating a body's move from advisory (C0-C3) to enforced (C4+) coordination, or verifying IT-018 Broker Collapse compliance. NOT for the inbound trigger→spawn boundary that decides whether an event may spawn an agent at all (use fleet-event-spawn-trust), the capability-token cryptographic mechanism behind a denial (use agentic-zero-trust-security), designing how a swarm of agents invokes and shards work (use swarm-invocation-designer), or auditing an individual PR's mergeability (use agent-pr-authoring).

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Design decisions for when and what sounds to use in web apps, mobile apps, and desktop native apps — plus how to create them with AI. Covers UI sound vocabulary (confirms, alerts, transitions, errors), platform conventions (iOS, macOS, Material), creating sounds with AI tools (ElevenLabs, AudioCraft, Stable Audio), Web Audio API for procedural sounds, haptic-audio pairing, and the psychology of satisfying vs. annoying UI sounds. Activate on 'app sounds', 'UI sounds', 'notification sound', 'sound design app', 'earcons', 'sonic branding', 'interaction sounds', 'feedback sounds', 'app audio', 'satisfying sounds'. NOT for professional audio engineering (use sound-engineer), Windows 3.1 retro sounds specifically (use win31-audio-design), music composition (use sound-engineer).

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Refactor apps so backend owns runtime truth and UI only projects snapshots. Use when tools have split authority. NOT for cosmetic UI work or new features before drift is removed.

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