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Jean — Molecular Biology & Genomics

design_hcr_probes

Read-only

Design HCR v3.0 split-initiator probe pairs against the subject: tile its reverse complement into 52 nt windows, drop windows with C or G runs, a stable hairpin, G+C or RNA:DNA free energy outside their windows, or (given a bowtie2 alignment of the tiles) more than one genome hit, then pick non-overlapping tiles closest to the target free energy and split each around a 2 nt gap into an odd and an even probe carrying half of the chosen amplifier's initiator.

Input Schema

NameRequiredDescriptionDefault
_filesNoThe project: record references shaped like _subject. Their records become library documents a tool names in sequences/parts/document; a trace or alignment is found by file name.
max_gcNohighest percent G+C of a tile. In %.
min_gcNolowest percent G+C of a tile. In %.
channelNoHCR amplifier whose split initiator the pair carries; one channel per target in a multiplexed experiment.B1
dtm_maxNolargest allowed difference between the halves' melting temperatures. In °C. Only applies when dtm_filter is true. Advanced.
_subjectNoThe record to act on: exactly one of sequence, accession (+source), sample (an id from list_samples) or file ({name, text} or {name, base64}); record_index picks one record of several.
max_gibbsNoleast stable RNA:DNA free energy to accept. In kcal/mol.
min_gibbsNomost stable RNA:DNA free energy to accept (at 37 °C, 0.33 M Na⁺). In kcal/mol.
tile_sizeNolength of the target window each pair covers, both halves and the 2 nt gap. In nt. Advanced.
dtm_filterNoalso require the two halves' melting temperatures to agree. Advanced.
max_probesNomost probe pairs to return.
target_gibbsNofree energy the selection ranks tiles by closeness to. In kcal/mol.
alignment_samNoSAM from bowtie2 -k 3 -f of the tiles FASTA this tool returns, against your genome or transcriptome. Without it no tile is checked for uniqueness and the result says so.
max_hairpin_tmNodrop a tile whose primer3 hairpin melts at or above this. In °C. Advanced.
max_run_lengthNowindow length for the C and G run check. In nt. Advanced.
num_hits_allowedNomost alignments a tile may have and still be kept (its own locus counts as one). Only applies when alignment_sam is set.
max_run_mismatchesNobases in that window allowed to differ from the run base. In nt. Advanced.

Output Schema

NameRequiredDescriptionDefault
pairsYesthe selected probe pairs, ranked by closeness to the target free energy, each with both oligos, its 1-based start, G+C, Tm, half-Tm difference, free energy and genome hits
stagesYeshow many tiles each stage kept, in pipeline order
pairCountYeshow many probe pairs were selected
orderTableYesevery odd then every even oligo as a Name/Sequence table for plate ordering
tilesFastaYesthe tiles that pass the run and hairpin checks, as FASTA, for bowtie2 -k 3 -f
explanationYesthe derivation chain: conclusion, interpretation and machinery, generated from this run

Schema Changelog

Changes observed during successful MCP inspections.

  1. First observed

TDQS

A4.2/5.0
Behavior4/5

Does the description disclose side effects, auth requirements, rate limits, or destructive behavior?

Annotations already declare readOnlyHint/openWorldHint=false, so the safety profile is covered; the description goes further by disclosing the rejection criteria (C/G runs, hairpin, G+C and RNA:DNA free-energy windows, genome hits), the non-overlap and ranking rule, and notably what happens when alignment_sam is omitted and that the result self-reports it. That conditional-degradation note is genuinely useful behavior beyond the annotations. It stops short of stating runtime cost or output volume, so a 4 rather than a 5.

Agents need to know what a tool does to the world before calling it. Descriptions should go beyond structured annotations to explain consequences.

Conciseness4/5

Is the description appropriately sized, front-loaded, and free of redundancy?

Purpose is front-loaded in the first four words, then the colon-and-list structure walks the pipeline in execution order with no filler sentences. The single sentence is long and dense enough that an agent must parse a nested list, which keeps it off a 5.

Shorter descriptions cost fewer tokens and are easier for agents to parse. Every sentence should earn its place.

Completeness5/5

Given the tool's complexity, does the description cover enough for an agent to succeed on first attempt?

For a 17-parameter, nested-object tool with an output schema and read-only annotations, the description covers the algorithm end to end plus the one behavioral fork that matters (missing alignment). The _subject resolution rules and channel semantics are already documented in the schema, so nothing an agent needs before invoking is absent.

Complex tools with many parameters or behaviors need more documentation. Simple tools need less. This dimension scales expectations accordingly.

Parameters4/5

Does the description clarify parameter syntax, constraints, interactions, or defaults beyond what the schema provides?

Schema coverage is 100%, so the baseline is 3, but the description ties the parameters into a coherent narrative — 52 nt tiling, 2 nt gap, run/hairpin/free-energy filters, target free energy for ranking, amplifier initiator — which tells the agent how the knobs interact rather than just what each means. That is meaning added on top of the per-parameter descriptions.

Input schemas describe structure but not intent. Descriptions should explain non-obvious parameter relationships and valid value ranges.

Purpose5/5

Does the description clearly state what the tool does and how it differs from similar tools?

Names a specific verb and resource — 'Design HCR v3.0 split-initiator probe pairs' — and then spells out the exact operation: tiling the reverse complement, filtering, ranking, and splitting around a 2 nt gap. An agent can distinguish this from design_ish_probes, design_merfish_probes, or design_xenium_probes on the HCR v3.0 split-initiator specificity alone, without opening any schema.

Agents choose between tools based on descriptions. A clear purpose with a specific verb and resource helps agents select the right tool.

Usage Guidelines3/5

Does the description explain when to use this tool, when not to, or what alternatives exist?

The description explains the pipeline's internal mechanics but never states when to choose this tool over a sibling probe-design tool, nor any prerequisites or exclusions. The only conditional guidance is parameter-level (alignment_sam: 'Without it no tile is checked for uniqueness and the result says so') and channel multiplexing. Usage is implied by the tool name rather than argued.

Agents often have multiple tools that could apply. Explicit usage guidance like "use X instead of Y when Z" prevents misuse.

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