dnasp

name: dnaspdescription: Population genetics of pre-aligned DNA sequences or multi-sample VCFs  using selected DnaSP 6 methods. Use for diversity, neutrality statistics, linkage  disequilibrium, InDel polymorphism, divergence, MK, Ka/Ks and codon usage; not alignment,  phasing or clinical interpretation.license: MITmetadata:   version: 0.6.1  author: David De Lorenzo  domain: molecular-evolution  tags:   - population-genetics  - molecular-evolution  - DNA-polymorphism  - neutrality-tests  - linkage-disequilibrium  - recombination  - divergence  - sequence-analysis  inputs:   - name: alignment    type: file    format:     - fasta    - fas    - nexus    - nex    description: Aligned DNA sequences (pre-aligned, equal-length). FASTA (including      DnaSP-style >'name' [comment] headers) or NEXUS (MATCHCHAR, INTERLEAVE).    required: false    cli_flag: --input  - name: vcf    type: file    format:     - vcf    description: Multi-sample VCF (--vcf). Converted to one aligned MSA per CHROM      (biallelic SNPs only; phased -> haplotype rows). Alternative to --input. Optional      --region CHROM, --vcf-merge to pool all CHROMs.    required: false    cli_flag: --vcf  - name: alignment2    type: file    format:     - fasta    - fas    - nexus    - nex    description: Second-population alignment for divergence analysis (--input2). Alternative      to --pop-file. Sequences must have same length as --input.    required: false    cli_flag: --input2  - name: pop_file    type: file    format:     - tsv    - txt    description: 'Population assignment file: one row per sequence, tab-separated      (sequence_name<TAB>population_name). Required for Fst; alternative to --input2 for divergence.'    required: false    cli_flag: --pop-file  - name: outgroup    type: string    description: Unique sequence identifier removed from the ingroup. Required for      fuliout, mk and faywu; also polarises SFS/TsTv and selects ingroup-versus-outgroup      Ka/Ks.    required: false    cli_flag: --outgroup  - name: hka_file    type: file    format:     - tsv    - txt    description: 'HKA locus file: whitespace-separated, exactly two loci, columns      locus n S L_poly D [L_div] [chrom]. Required for --analysis hka.'    required: false    cli_flag: --hka-file  - name: analyses    type: string    description: Comma-separated polymorphism, ld, recombination, popsize, indel,      divergence, fuliout, hka, mk, kaks, fufs, sfs, tstv, codon, faywu, fst; or all.      Polymorphism runs with alignment input.    required: false    cli_flag: --analysis  - name: window_size    type: integer    description: Sliding window size in bp (0 = whole alignment only, default 0)    required: false    cli_flag: --window  - name: step_size    type: integer    description: Sliding window step in bp (default = window_size)    required: false    cli_flag: --step  - name: genetic_code    type: string    description: 'Codon table for mk/kaks/codon: "standard" or "vertebrate-mitochondrial"      (TGA=Trp, AGA/AGG=stop, ATA=Met; for COII/cytb/ND-type loci). Default: standard.'    required: false    cli_flag: --genetic-code  - name: n_sim    type: integer    description: Coalescent replicates for whole-region P-values of Tajima's D, R2      and Fu's Fs (0 = no simulation, default). 10000 is typical.    required: false    cli_flag: --n-sim  - name: sim_given    type: string    description: 'Simulation conditioned on the observed segregating sites ("S",      default) or on Watterson''s theta ("theta").'    required: false    cli_flag: --sim-given  - name: sim_seed    type: integer    description: Seed for --n-sim; generated and recorded when omitted.    required: false    cli_flag: --sim-seed  outputs:   - name: report    type: file    format:     - md    description: Markdown analysis report with statistics and interpretation  - name: results_table    type: file    format:     - tsv    description: Polymorphism and sliding-window TSV only; other modules appear in      report.md.  - name: result    type: file    format:     - json    description: ClawBio result.json envelope (skill, version, input checksum,      headline summary, the summary.json payload plus artifact list,      chat_summary_lines and preferred_artifacts for the runner). A multi-CHROM      VCF run writes one envelope per CHROM directory and a root envelope that      summarises them with relative paths.  - name: summary    type: file    format:     - json    description: Module summaries (global and per-window statistics with DnaSP      midpoints, LD/recombination/mismatch/InDel/divergence/MK/Ka-Ks/codon results      including named per-sequence ENC); LD pair grids are in ld_pairs, not here.  - name: ld_pairs    type: file    format:     - tsv    description: Pairwise LD table (only when --analysis ld is active)  - name: figures    type: directory    description: Sliding-window plots, LD decay scatter, mismatch histogram (PNG)  - name: reproducibility    type: directory    description: Archived inputs, commands.sh, environment.yml, manifest.json and      output-relative checksums.sha256.  dependencies:     python: '>=3.10'    packages:     - matplotlib>=3.7    - numpy>=1.24    - pandas>=2.0    - opentelemetry-sdk>=1.20  demo_data:   - path: examples/demo_simple.fas    description: Synthetic 6-sequence × 10-bp alignment with known statistics  - path: examples/demo_rp49.fas    description: rp49 region, 17 Drosophila sequences, 300 bp  endpoints:     cli: python skills/dnasp/dnasp.py --input {alignment} --analysis {analyses} --output      {output_dir}  openclaw:     requires:       bins:       - python3    always: false    emoji: ''    homepage: https://github.com/ClawBio/ClawBio    os:     - darwin    - linux    - win32    install:     - kind: pip      package: matplotlib>=3.7    - kind: pip      package: numpy>=1.24    - kind: pip      package: pandas>=2.0    - kind: pip      package: opentelemetry-sdk>=1.20    trigger_keywords:     - nucleotide diversity    - Tajima's D    - DNA polymorphism    - population genetics sequences    - haplotype diversity    - DnaSP    - segregating sites    - Fu and Li test    - neutrality test alignment    - Watterson theta    - linkage disequilibrium    - recombination events    - mismatch distribution    - population expansion    - InDel polymorphism    - divergence between populations    - Dxy Da net divergence    - fixed differences populations    - Ramos-Onsins Rozas R2    - Fu Li D F outgroup    - outgroup polarised mutations    - HKA test neutrality    - Hudson Kreitman Aguade    - two-locus neutrality    - polymorphism divergence ratio    - McDonald-Kreitman test    - MK test    - adaptive evolution test    - alpha McDonald-Kreitman    - neutrality index NI    - direction of selection DoS    - Ka/Ks    - dN/dS    - omega synonymous nonsynonymous    - synonymous substitution rate    - nonsynonymous substitution rate    - coding sequence neutrality    - Nei-Gojobori method    - Fu's Fs test    - Fu 1997 Fs    - site frequency spectrum    - SFS folded unfolded    - allele frequency spectrum    - singleton excess    - minor allele frequency distribution    - VCF population genetics    - multi-sample VCF nucleotide diversity    - VCF to haplotypes    - population genomics from VCF

DnaSP

Trigger

Fire when a user requests population-genetic analysis of aligned DNA, a supported VCF, or a DnaSP-compatible statistic listed below. Do NOT fire for sequence alignment, read mapping, haplotype phasing, clinical advice, or significance tests other than the coalescent P-values --n-sim gives for D, R2 and Fs.

Scope

Analyse genetic variation in supplied alignments using 16 selected DnaSP methods. This is not a complete replacement for the DnaSP GUI or all its analysis modes. Read the statistical reference for definitions, exclusions, source conventions, file formats, examples and release validation evidence.

Core Capabilities

• Polymorphism: S, Eta, haplotypes, Hd, VarHd, pi, k, theta-W, G+C, Tajima's D, Fu and Li D*/F*, Ramos-Onsins and Rozas R2.
• LD: D, D', R2, ZnS, Za, ZZ and original-column pair labels; recombination Rm.
• Mismatch distribution with unbiased variance over unordered pairs and Sokal & Rohlf corrected CV; Harpending (1994) raggedness; Model 1 diallelic InDel diversity.
• Divergence and Hudson Fst between populations; outgroup Fu and Li D/F.
• Two-locus HKA, McDonald-Kreitman, Nei-Gojobori Ka/Ks, Fu's Fs and SFS.
• Opt-in coalescent P-values (--n-sim) for Tajima's D, R2 and Fu's Fs under a null with no recombination. A statistic whose simulated null takes one value (R2 with two sequences; Fu's Fs with two sequences given S; Tajima's D below four, its variance being zero at three) is reported without a P-value. A seed replays only on the same Python.
• Ts/Tv, codon counts/RSCU including stops, named per-sequence ENC and its synonymous-codon-weighted summary.
• Sliding windows mirror DnaSP's Gaps in Sliding Window = considered mode: starts advance from 1 by the step, capped at alignment length; ends are capped there too, and the first window reaching the end terminates the loop. Empty windows are retained. The not-considered mode is not implemented.
• Window midpoints are the alignment position of the ceil(net/2)-th gap-free column, or the window start if none exists; exported in TSV, JSON and report and used for the plot. VCF windows use retained SNP indices, including the final partial window.
• Raw per-site Fay-Wu H and theta-L minus theta-W; normalised Hn/ZE are absent.

Fu and Li D*/F* and outgroup D/F mirror Data > Segregating Sites/Mutations = Segregating sites, using DnaSP's v5-style panels. The rp49 Eta-setting D*/F* figures can be reproduced by substituting eta for S, but this is not a general conversion: FULI.vb also changes singleton and external-mutation capping (SingleMut and ExternaMut subtraction). No Eta-mode switch is implemented.

Workflow

1. Confirm the input is pre-aligned and identify the scientific comparison: ingroup, explicit outgroup, populations, coding interval and genetic code. Do not infer an outgroup from record order or silently select a code.
2. Use the full repository checkout or supplied validation code snapshot so the shared reproducibility helpers are available. Install the declared dependencies.
3. Choose an empty output directory. For alignment input use --input; for VCF use --vcf; for two-locus HKA alone use --hka-file --analysis hka.
4. Select actual implemented names with --analysis. Supply --outgroup, --input2 or --pop-file when needed. Use --genetic-code vertebrate-mitochondrial for the matching mitochondrial table. The default is standard. Coding intervals must be preselected and divisible by three.
5. Execute the CLI. An explicit analysis that cannot run returns a non-zero code. --analysis all is opportunistic: inspect its completed/skipped manifest.
6. Read stderr diagnostics, report.md and reproducibility/manifest.json. Distinguish a failed analysis, an undefined statistic and an excluded site.
7. Interpret the chosen statistic within its documented assumptions. Do not turn a signed value or a threshold into a significance claim; only --n-sim P-values assess significance, and a rejection does not by itself identify its cause.
8. Retain the input archive, settings, hashes and environment with the report. commands.sh replays on the recorded host/code path into a new output folder; moving a run requires the code and dependencies as well as its input archive.
9. For Windows GUI comparison, follow the separate validation checklist and capture raw DnaSP output. Source-derived expectations are not GUI observations.

Example Output

python skills/dnasp/dnasp.py --demo --output new_demo_run
python skills/dnasp/dnasp.py --input alignment.fas --analysis polymorphism,ld --output new_run
python skills/dnasp/dnasp.py --input coding.fas --outgroup OutSeq --genetic-code vertebrate-mitochondrial --analysis mk,kaks,codon --output new_coding_run
python skills/dnasp/dnasp.py --vcf samples.vcf --analysis polymorphism,sfs,fufs --output new_vcf_run

The synthetic demo contains 10 ingroup sequences, one outgroup and 300 sites:

The demo runs 15 modules; HKA uses a separate two-locus input. The regression suite, rather than the printed banner alone, checks the expected figures.

Output Structure

output/
  report.md
  results.tsv                  # polymorphism and windows
  summary.json                 # module summaries, window midpoints, named ENC/null
  result.json                  # ClawBio envelope: headline summary, summary.json payload, artifacts
  ld_pairs.tsv                 # when LD pairs exist
  figures/                     # when matplotlib is available
  reproducibility/
    inputs/
    commands.sh
    environment.yml
    manifest.json
    checksums.sha256

Dependencies

Python 3.10 or later. Core estimators use the standard library. Plotting uses matplotlib; the repository's shared reproducibility package also imports NumPy, pandas and OpenTelemetry. Use environment.yml or the validation package's pinned requirements. The Windows validation runner targets Python 3.12.

Gotchas

• The model will want to count IUPAC symbols as alleles. Do not. Supported ambiguity symbols are missing data and the nucleotide mask excludes their columns.
• The model will want to analyse an unknown outgroup as an ingroup-only run. Do not. Identifiers must be unique and an explicit outgroup must resolve.
• The model will want to call VCF diversity per-base diversity. Do not. VCF columns represent retained variant records; invariant callable bases are absent.
• The model will want to use any coding annotation in a NEXUS file. Do not. This implementation requires a preselected coding alignment and does not read CHARSET coding annotations. In COII, use positions 1-681 for codon usage.
• The model will want to apply one stop-codon rule everywhere. Do not. Selected stops count in RSCU and as family 21 in MK/KaKs; ENC and coding G+C use sense codons.
• The model will want to read raw H/E as normalised DnaSP Hn/ZE. Do not. These outputs are explicitly different, and no significance test is supplied.
• The model will want to overwrite an earlier output folder. Do not. Choose a fresh folder; runs reject non-empty destinations to preserve prior artefacts.
• The model will want to infer genomic-scale performance from the tiny bundled VCF examples. Do not. LD enumerates all biallelic-site pairs and uses quadratic storage; read the measured limits in the reference before a large run.

Known Differences from DnaSP 6.12.03

Four values compared with DnaSP 6.12.03 are unresolved. They come from the DnaSP VCF examples with a single variant site: the phased diploid Scaffold_2 (n = 20) and the haploid Region_MSA_2 (n = 10). The skill follows the DnaSP 6 source code and the result files shipped with DnaSP 6.0.60; the captures cannot tell whether DnaSP's build or its analysis route changed.

Differences of setting or definition, not errors:

• Fu and Li's tests follow DnaSP's Segregating sites setting; DnaSP's default Eta setting gives different D*, F*, D and F.
• DnaSP prints two F* forms; the skill reproduces the "DnaSP v5" form (Simonsen et al. 1995), not the biallelic-positions form (Achaz 2009) that DnaSP's multi-alignment output reports.
• A coding region whose final codon is a stop in every sequence is analysed without that codon; DnaSP keeps it if the user declines its prompt.
• The folded SFS excludes multiallelic sites; DnaSP's segregating-sites spectrum places them in a frequency class.
• Sliding windows with no segregating site report Tajima's D as undefined; DnaSP prints 0.0000.
• Coalescent simulation (--n-sim): the null has no recombination, so for a recombining region a low Fu's Fs P-value may reflect recombination rather than growth (Tajima's D and R2 are conservative there). DnaSP 6 offers a recombination setting; DnaSP 6's own one-locus module conditions on theta only, fixed S being its v5 routine; and DnaSP reports the one-tailed P(Sim <= Obs), where this skill reports twice the smaller tail for Tajima's D.
• Ts/Tv has no DnaSP 6 counterpart, and raw Fay and Wu H and Zeng E are not DnaSP's normalised Hn and ZE.

Version History

Current version 0.6.1. Every change that alters a result, and the version compared with DnaSP 6.12.03 (0.5.2), is recorded in docs/version_history.md.

Safety

All sequence analysis and output remain local. ClawBio is a research and educational tool. It is not a medical device and does not provide clinical diagnoses. Consult a healthcare professional before making any medical decisions.

Agent Boundary

The agent selects documented inputs/options, executes the skill and explains reported results. The code computes the statistics. Neither the agent nor the skill may fabricate GUI validation, P-values or missing results.

Integration with Bio Orchestrator

CLI alias: dnasp. Use --analysis for module selection, not invented flags such as --pi, --kaks or --tajima. The repository dispatcher permits the implemented options, including VCF, populations and genetic-code selection.

Route here when the user asks for a statistic this skill computes (for example nucleotide or haplotype diversity, Tajima's D, Fu and Li's tests, Fu's Fs, McDonald-Kreitman, Ka/Ks, HKA, the mismatch distribution, InDel polymorphism or codon usage bias) on an aligned FASTA or NEXUS file or a multi-sample VCF. INTENTS.json publishes the dnasp aliases to ClawBio's intent planner and plans the demo only when the user explicitly asks for a demo; a real analysis needs the user's file, and --analysis selects modules other than the default polymorphism summary.

Chaining Partners

• Alignment upstream. Sequences must be aligned before this skill. phylogenetics-builder aligns with MAFFT, MUSCLE and other aligners. Versions of that skill that save their alignment write alignment/aligned.fasta; use that untrimmed file rather than the trimAl output, because trimming removes gapped or poorly aligned columns and so changes S, pi and the InDel results. Otherwise align the sequences separately and pass the aligned FASTA.
• fastreer. Builds distance trees from the same multi-sample VCF: run this skill for diversity and neutrality statistics, then fastreeR for sample relationships.
• claw-ancestry-pca. Gives population-structure context before samples are grouped in a --pop-file for Fst or divergence.
• equity-scorer. Reports HEIM heterozygosity and Fst from VCF or ancestry data with its own estimators; this skill's Hudson Fst and diversity statistics follow DnaSP 6, so the two sets of values are not interchangeable.
• VCF preparation. A filtering or phasing workflow may prepare input, but every filter and phasing choice must be recorded; unphased heterozygotes stay unresolved here.
• Reporting downstream. Use report.md, summary.json, result.json and the TSV files; the TSV does not contain every module's results.

Maintenance

Recheck source/help-derived regression cases and the 170 historical comparison fixtures after changes to formulas, masks or parsers. Review GUI differences when the target DnaSP build or analysis mode changes. Keep this file, the method reference, CLI metadata, version and catalogue consistent. New Windows GUI observations must be reviewed before changing published concordance counts.