design_qpcr_assay
Design qPCR assays over the subject — primer pair plus hydrolysis probe, ranked by penalty, with amplicon size/GC, per-oligo Tm and whether both primers bind a single site.
Input Schema
TableJSON Schema
| Name | Required | Description | Default |
|---|---|---|---|
| _files | No | The 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. | |
| _subject | No | The 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. | |
| conditions | No | salt, magnesium, dNTP and oligo concentrations plus the salt-correction method and any DMSO or formamide that every Tm in this design is measured in. Left unset it is filled from your reaction-conditions settings, and falls back to the nearest-neighbour defaults when there are none. Advanced. | |
| max_poly_x | No | longest run of one base allowed in any oligo (primer3's POLY_X). A long run mispairs by slipping, and for a TaqMan probe a run of four or more Gs quenches the reporter. In nt. Advanced. | |
| region_end | No | 0-based inclusive end. Unset = the whole sequence. Advanced. | |
| max_results | No | how many assays to return, best penalty first. | |
| amplicon_max | No | longest acceptable amplicon - qPCR amplicons are short on purpose, because a short product amplifies efficiently enough for the exponential phase to be clean. In bp. | |
| amplicon_min | No | shortest acceptable amplicon. In bp. | |
| probe_max_gc | No | highest acceptable probe GC content. In %. Advanced. | |
| probe_max_tm | No | probes above this Tm are rejected outright. In °C. Advanced. | |
| probe_min_gc | No | lowest acceptable probe GC content. In %. Advanced. | |
| probe_min_tm | No | probes below this Tm are rejected outright. In °C. Advanced. | |
| probe_tm_opt | No | target probe Tm - conventionally ~10 °C above the primers, so the probe is bound before extension reaches it. In °C. | |
| region_start | No | 0-based inclusive start of the region to design in. Unset = the whole sequence; pass the selection's start to scope to it. Advanced. | |
| primer_max_gc | No | highest acceptable primer GC content. In %. Advanced. | |
| primer_max_tm | No | primers above this Tm are rejected outright. In °C. Advanced. | |
| primer_min_gc | No | lowest acceptable primer GC content. In %. Advanced. | |
| primer_min_tm | No | primers below this Tm are rejected outright, not merely penalised. In °C. Advanced. | |
| primer_tm_opt | No | target primer Tm; the penalty ranks candidates by their distance from it. In °C. | |
| probe_chemistry | No | what the hydrolysis probe is made of. `dna` is unmodified DNA; `lna` locks the bases named by `probe_locked_positions`, which raises the probe Tm and is how a short probe reaches the TaqMan offset over its primers. MGB and Scorpion chemistries are not offered: neither has a published nearest-neighbour parameter set to compute a Tm from. | dna |
| structure_check | No | run the `thal` secondary-structure check on the RETURNED assays: hairpin and cross-dimer melting temperatures for all three oligos and every pairing between them. On by default, because a qPCR assay that dimerises is the commonest design failure and penalty ranking alone will happily put one first. Off returns null structures and is measurably faster - the check is a dynamic-programming alignment per oligo pair. Advanced. | |
| probe_max_length | No | longest probe considered. In nt. Advanced. | |
| probe_min_length | No | shortest probe considered. In nt. Advanced. | |
| probe_opt_length | No | probe length the penalty prefers. In nt. Advanced. | |
| primer_max_length | No | longest primer considered; primer3's own ceiling is 36. In nt. Advanced. | |
| primer_min_length | No | shortest primer considered. In nt. Advanced. | |
| primer_opt_length | No | primer length the penalty prefers. In nt. Advanced. | |
| probe_tm_offset_min | No | how many °C the probe Tm must exceed the MEAN primer Tm. TaqMan guidance is 8-10; below that the probe can melt off before it is cleaved. In °C. | |
| probe_locked_positions | No | which bases to lock, 1-based from the probe's 5′ end, e.g. `3, 6, 9`. Read only when `probe_chemistry` is `lna`. Positions must be interior to the SHORTEST probe the search will try and never adjacent, because McTigue et al. (2004) measured one locked base per nearest neighbour and none at a duplex end - a position that breaks either rule is refused by name rather than silently dropped. Only applies when probe_chemistry is lna. |
Output Schema
TableJSON Schema
| Name | Required | Description | Default |
|---|---|---|---|
| best | Yes | the best-penalty assay | |
| count | Yes | how many assays were found | |
| probe | Yes | the best assay's probe | |
| assays | Yes | the ranked assays | |
| offset | Yes | where the designed-in stretch starts in the record: add it to every position above for record coordinates. 0 unless a region was given | |
| forward | Yes | the best assay's forward primer | |
| reverse | Yes | the best assay's reverse primer | |
| conditions | Yes | the buffer every Tm in this design was measured in - salt, magnesium, dNTPs, oligo concentration, the salt-correction method and any additives - or a note saying none were supplied | |
| probe_ordered | Yes | the best assay's probe as it must be ORDERED: plain DNA, or the locked-base notation (`GTCGLAAC`) when the probe was designed as LNA |