design_bisulfite_primers
Design BSP or MSP primers for a methylation assay, on the CONVERTED strands: after bisulfite treatment every unmethylated C reads as T, which is a different template from the one every other designer sees. BSP primers avoid CpGs so they amplify regardless of methylation state; MSP primers sit ON CpGs so they discriminate. Also reports the CpG islands (EMBOSS cpgplot) the assay should target.
Input Schema
TableJSON Schema
| Name | Required | Description | Default |
|---|---|---|---|
| assay | No | BSP = bisulfite sequencing PCR: primers avoid CpG sites so both methylated and unmethylated template amplify equally, and the methylation is read from the SEQUENCE afterwards. MSP = methylation-specific PCR: primers cover CpG sites so amplification ITSELF is the readout. They answer different questions and are not interchangeable. Values: BSP (BSP - amplify regardless of methylation), MSP (MSP - amplify only one state). | BSP |
| _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. | |
| max_gc | No | highest acceptable GC. In %. Advanced. | |
| max_tm | No | primers above this Tm are rejected outright. In °C. Advanced. | |
| min_gc | No | lowest acceptable GC. The floor is low on purpose: after conversion every unmethylated C is a T, so converted DNA is genuinely GC-poor and an ordinary 40% floor rejects correct primers. In %. Advanced. | |
| min_tm | No | primers below this Tm are rejected outright. In °C. Advanced. | |
| opt_tm | No | target Tm; candidates rank by distance from it. Bisulfite-converted DNA is C-poor, so these run lower than an ordinary design. In °C. | |
| min_cpg | No | MSP ONLY. The fewest CpG sites a primer must cover, so it can discriminate methylated from unmethylated at all. In sites. | |
| _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. | |
| criteria | No | which published criteria call a CpG island: Gardiner-Garden & Frommer 1987 as EMBOSS cpgplot encodes it, or the stricter Takai & Jones 2002, which excludes most Alu repeats. Values: cpgplot (Gardiner-Garden & Frommer (cpgplot)), takaiJones (Takai & Jones 2002 (promoter islands)). | cpgplot |
| max_length | No | longest primer considered. In nt. Advanced. | |
| min_length | No | shortest primer considered. In nt. Advanced. | |
| opt_length | No | primer length the ranking prefers. In nt. | |
| region_end | No | 0-based inclusive end. Unset = the whole sequence. Advanced. | |
| max_results | No | how many primer pairs to return, best first. | |
| product_max | No | longest acceptable amplicon. In bp. | |
| product_min | No | shortest acceptable amplicon. In bp. | |
| 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. | |
| island_window | No | sliding window over which C, G and CpG are counted (cpgplot -window); unset takes the preset's. In bp. Advanced. | |
| min_non_cpg_c | No | BSP ONLY. The fewest non-CpG cytosines the ORIGINAL sequence under a primer must carry - the rule that makes a BSP primer bisulfite-SPECIFIC, because those cytosines become T only if conversion worked. A primer with none anneals to untreated DNA just as well and the assay silently reports on template that was never converted. In nt. | |
| design_in_island | No | design inside this island only, by its rank in the reported list (1 = first). Unset designs anywhere in the region; the panel instead asks for an island to be picked. | |
| island_min_length | No | minimum island length (cpgplot -minlen); unset takes the preset's. In bp. Advanced. | |
| island_min_obs_exp | No | minimum observed/expected CpG (cpgplot -minoe); unset takes the preset's. Advanced. | |
| island_min_percent | No | minimum %C+%G (cpgplot -minpc); unset takes the preset's. In %. Advanced. | |
| max_cpg_distance_from_3 | No | MSP ONLY. How close to the 3' end the last CpG must sit. A mismatch discriminates most strongly at the 3' end, because that is where the polymerase extends. In nt. Advanced. |
Output Schema
TableJSON Schema
| Name | Required | Description | Default |
|---|---|---|---|
| assay | Yes | which assay was designed, echoed so a result never has to be guessed at | |
| count | Yes | how many pairs were found | |
| pairs | Yes | the primer pairs, best first: each with both oligos, their coordinates on the ORIGINAL unconverted sequence, Tm, GC, CpG count and the non-CpG C conversion check | |
| forward | Yes | the best pair's forward primer (wire into pcr_amplify) | |
| islands | Yes | CpG islands in the record (EMBOSS cpgplot criteria) - what a methylation assay should be aimed at | |
| reverse | Yes | the best pair's reverse primer | |
| islandCount | Yes | how many CpG islands were found | |
| methylatedPairs | Yes | MSP: the pairs specific for the METHYLATED allele. Empty under BSP | |
| unmethylatedPairs | Yes | MSP: the pairs specific for the UNMETHYLATED allele - the other half of the assay, and the control that tells a negative from a failed reaction. Empty under BSP |