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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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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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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.

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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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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).

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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.

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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.

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Build forest plots (HR, OR, RR, beta-coefficient summaries with CIs) and funnel plots (meta-analysis publication-bias diagnostics) using forestplot, metafor, ggforest, and MendelianRandomization with proper axis-scaling, summary-diamond placement, subgroup nesting, and Egger / trim-and-fill asymmetry tests. Use when summarizing effects across subgroups, trials, or instruments — meta-analysis, Mendelian randomization, subgroup HRs.

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Download large datasets from NCBI efficiently using EPost, history server, batching, rate limiting, and retry logic. Use when bulk-fetching tens of thousands of sequences, pulling all results of a large ESearch, designing reproducible pipelines, comparing E-utilities to NCBI Datasets v2 CLI, or implementing checksum-validated downloads. Encodes WebEnv TTL (~8h), EPost 200-ID limit, retmax caps, parallelization design, and integrity verification.

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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 publication-quality figures with matplotlib using the object-oriented Figure/Axes API, constrained_layout, rcParams customization, TrueType (Type-42) font embedding for journal submission, and CVD-safe palettes. Covers seaborn integration, common chart types, axis formatting, and the small gotchas that distinguish reproducible matplotlib from notebook scratch. Use when producing publication figures in Python — RNA-seq scatter, single-cell embeddings, generic biological plotting.

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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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Detect distant homologs using profile and structure-aware methods that go beyond standard BLAST. Use when sequence identity falls into the twilight zone (<35% pairwise), when BLAST fails to find homologs that should exist, when working at metagenomic scale (DIAMOND, MMseqs2), or when structure beats sequence (Foldseek). Covers PSI-BLAST (iterative PSSM), jackhmmer (iterative HMM), HHblits/HHsearch (profile-profile), DIAMOND, MMseqs2, and Foldseek (3Di structural alphabet, van Kempen 2024).

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Build local BLAST databases and run searches using NCBI BLAST+ command-line tools. Use when running >50 queries, building custom databases with -parse_seqids and -taxid, downloading prebuilt NCBI databases via update_blastdb.pl, choosing -task variants (megablast/dc-megablast/blastn/blastn-short), tuning soft/hard masking, scaling threads, or extracting hits with blastdbcmd. Encodes BLAST v5 vs v4 database format, taxonomy filtering, makeblastdb pitfalls.

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Select colormaps and qualitative palettes for scientific figures using perceptual-uniformity, color-vision-deficiency safety, and luminance-monotonicity criteria. Covers Crameri scientific colormaps, viridis/cividis/magma, Okabe-Ito categorical, ColorBrewer, and the rainbow/jet critique. Use when choosing palettes for heatmaps, scatter, networks, or any encoding where color carries quantitative or categorical meaning.

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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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Retrieve records from NCBI databases using Biopython Bio.Entrez (EFetch, ESummary). Use when downloading sequences, fetching GenBank/GenPept records, getting document summaries, parsing nested XML, navigating GI deprecation, choosing between rettype+retmode combinations, and parsing into Biopython SeqRecord/SwissProt objects. Covers nucleotide, protein, gene, pubmed, sra, gds, taxonomy, snp, clinvar.

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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.

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Run remote BLAST searches against NCBI servers using Biopython Bio.Blast.NCBIWWW. Use when identifying unknown sequences, finding homologs, picking the correct BLAST program (blastn/blastp/blastx/tblastn/tblastx/psiblast/megablast/dc-megablast), interpreting Karlin-Altschul E-values, avoiding the max_target_seqs trap (Shah 2019), choosing composition-based statistics, or limiting searches by organism. Covers RID lifecycle, database choice (nt/nr/refseq_select/swissprot), word-size and CBS taxonomy.

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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.

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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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Runs JACKS (Joint Analysis of CRISPR/Cas9 Knockout Screens; Allen et al 2019 Genome Research) which models per-sgRNA log-fold-change as the product of a treatment-dependent gene-essentiality term and a treatment-independent guide-efficacy term. Covers the Bayesian decomposition math, the hierarchical efficacy prior shared across screens performed with the same library, when JACKS outperforms MAGeCK (multi-screen joint analysis, libraries with broad efficacy variance) and when it does not (single screen, novel libraries with no prior efficacy), library-reuse efficacy transfer, downstream essentiality interpretation, and the 2.5x sample-size reduction enabled by efficacy-aware testing. Use when running multiple screens with the same library, when guide-level noise is suspected to dominate per-gene signal, when reusing published essentiality reference screens for efficacy priors, or when comparing screens performed across cell lines that share library but differ biologically.

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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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Identify direct miRNA-target interactions from AGO HITS-CLIP, AGO-CLEAR-CLIP (chimeric reads), HEAP (Halo-Ago2 mouse), chimeric eCLIP / miR-eCLIP (deep miRNA-target profiling), or CLASH using chimeric-read processing pipelines, seed-pairing analysis, and 3' auxiliary pairing rules. Use when distinguishing direct miRNA targets from indirect, integrating CLIP-derived target maps with TargetScan / miRDB / DIANA predictions, applying canonical 7mer-8mer seed matching with 3' UTR context, or recovering miRNA-mRNA chimeras at scale.

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