design_merfish_probes
Read-only
Design MERFISH encoding probes for the subject transcript: tile it into target regions, keep those whose G+C and melting temperature fall in their windows, that share no k-mer with an rRNA or tRNA, and whose k-mers occur in this gene's transcripts and nowhere else in the transcriptome; attach readout sequences that spell the gene's codeword, add the amplification primers, screen the whole oligo again, select probes that cover every on-bit evenly with least overlap, and drop probes complementary to one another.
Input Schema
| Name | Required | Description | Default |
|---|---|---|---|
| seed | No | seed for the random draws in readout assignment and selection. Advanced. | |
| _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 G+C of a target region (exclusive). In %. | |
| max_tm | No | highest melting temperature of a target region (exclusive). In °C. | |
| min_gc | No | lowest G+C of a target region (exclusive). In %. | |
| min_tm | No | lowest melting temperature of a target region (exclusive). In °C. | |
| spacer | No | bases between each readout and the target. Advanced. | A |
| trials | No | selection runs; the one with the most even on-bit coverage is kept. Advanced. | |
| barcode | No | the gene's codeword, one 0 or 1 per bit, 1 where the bit is on. | |
| rtrna_k | No | k-mer length of the rRNA/tRNA screen. Advanced. | |
| _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. | |
| probe_nm | No | concentration of each probe. In nM. Advanced. | |
| readouts | No | the readout sequences as FASTA, one per bit, in bit order. | |
| formamide | No | formamide in the hybridization buffer. In %. Advanced. | |
| sodium_mm | No | monovalent salt of the hybridization buffer. In mM. Advanced. | |
| t7_length | No | bases of the reverse primer lost in in vitro transcription. In nt. Only applies when reverse_primer is set. Advanced. | |
| target_id | No | the transcript to design for, by its FASTA id. Empty: the transcript whose sequence is the subject's. Advanced. | |
| expression | No | expression per transcript, one 'id value' line each (FPKM or TPM). Empty: every transcript weighs 1, MERFISH_probe_design's own choice without expression data. Advanced. | |
| cross_talk_k | No | k-mer length of the library-wide cross-hybridization screen. Advanced. | |
| max_rtrna_ot | No | highest rRNA/tRNA off-target count to keep (exclusive); 0.5 keeps only regions with none. Advanced. | |
| specificity_k | No | k-mer length of the specificity and readout off-target counts. Advanced. | |
| target_length | No | length of each target region. In nt. | |
| transcriptome | No | every transcript of the organism as FASTA (RefSeq titles name the gene in their last parentheses); the subject is found in it by sequence. | |
| forward_primer | No | forward primer; its reverse complement ends the oligo. Only applies when reverse_primer is set. Advanced. | |
| reverse_primer | No | reverse primer, T7 promoter at its 5' end. Empty: no primers are added. Advanced. | |
| min_specificity | No | lowest specificity to keep (exclusive): the share of a region's k-mers found only in this gene. | |
| rtrna_reference | No | rRNA and tRNA sequences as FASTA. An Ensembl ncRNA FASTA is filtered to those biotypes by its headers. Empty: the rRNA/tRNA screen does not run. | |
| one_bit_per_probe | No | give each probe copies of a single readout. Advanced. | |
| readout_assignment | No | how each probe's on-bits are drawn: balanced keeps every on-bit equally used down the transcript. Advanced. | balanced |
| readouts_per_probe | No | readout sequences carried by each probe, split between its two ends. | |
| probes_per_transcript | No | probes to select. | |
| max_readout_ot_increase | No | drop a probe whose readouts add this many expression-weighted off-target k-mers or more (the single-molecule pipeline's check). Empty: not checked. Advanced. |
Output Schema
| Name | Required | Description | Default |
|---|---|---|---|
| probes | Yes | the selected encoding probes in selection order, each with its 0-based shift, target, G+C, Tm, specificity, isospecificity, readouts, barcode and the oligo to order | |
| report | Yes | the transcript-level report: probes, probes per on-bit, the fewest on any bit, and how many could be placed without overlap before and after selection | |
| stages | Yes | what each stage took in and kept, in pipeline order, with the reason a skipped stage did not run | |
| orderTable | Yes | every probe as a Name/Sequence table for pool ordering | |
| probeCount | Yes | how many encoding probes were kept | |
| explanation | Yes | the derivation chain: conclusion, interpretation and machinery, generated from this run |