In-Situ Probes
Design a hybridization probe against a transcript or a locus: tile it into oligos, screen them for off-target binding, and pick the set that images as one spot. Or design padlock probes for in-situ sequencing, checked for off-target ligation at the junction, or HCR v3.0 split-initiator probe pairs for amplified imaging.
| Reached by | Tools → In-Situ Probes |
| Formats | Any sequence already open in the project (GenBank, FASTA, SnapGene, .melad …) A SAM alignment of the candidates BLAST tabular output (padlock targets) FASTA and BED out (the oligos) FASTA out (padlock targets and probes) SAM in, FASTA and an IDT-style Name/Sequence table out (HCR pairs) |
What it does
An in-situ hybridization experiment makes one RNA or one locus visible inside a fixed cell by hybridizing many short oligos along it, each carrying a fluorophore or a readout sequence. What you see down the microscope is the sum of those oligos, so the design question is never "is this a good oligo" on its own. It is three questions, and they have to be answered in order.
Can each oligo hybridize? An oligo that melts off at the hybridization temperature contributes nothing, and one that binds far too tightly contributes background. So the target is walked window by window and each window is judged on length, melting temperature, G+C content, and whether it contains a run of one base — homopolymer runs hybridize promiscuously and are excluded by default.
Would any of them bind somewhere else? This is the question that separates a probe that works from one that images the wrong thing, and it cannot be answered from the oligo alone: it needs the rest of the genome. Align the candidates against your reference and the alignment tells you which oligos have a competitor. A trained linear discriminant then judges whether that competitor is thermodynamically real at your hybridization temperature, which recovers usable probes near repeats that a strict unique-alignment rule would throw away.
Do they image as one spot? A set spread thinly across a long transcript is a smear, not a spot, and a set clumped at one end images that end. So candidate sets of consecutive oligos are scored on three properties — how centred the set is on the molecule, how compact it is, and how evenly its oligos are spaced — and you choose which one defines the acceptable band and which ranks inside it.
What this computes
Per candidate oligo
| Readout | What it is |
|---|---|
| Melting temperature | Nearest-neighbour Tm under the unified SantaLucia parameters, salt-corrected for the sodium you specify, computed at the probe and target strand concentrations you set (25 nM each by default, as OligoMiner does), and then corrected for formamide. It is the temperature at the bench, not in water. |
| Percent G+C | Counted over the oligo, with S counted as a G or C base. |
| Length and coordinates | Where the oligo sits on the target, 1-based inclusive. When the target carries a range= header from a genome browser, the coordinates are the genome's. |
Per screening decision
| Readout | What it is |
|---|---|
| Off-target probability | The probability that an oligo's best competing site is thermodynamically real, from a linear discriminant over three features: the oligo's length, the alignment score of its best off-target hit, and its percent G+C. Six models exist, one per hybridization temperature (32, 37, 42, 47, 52 and 57 °C). |
| Verdict | Whether an oligo was kept because it aligned uniquely, because it aligned nowhere, or because the model judged its off-target site unlikely. |
Per probe set
| Readout | What it is |
|---|---|
| Centrality | Where the set's midpoint sits relative to the middle of the region: 1 when they coincide, 0 at either end, negative when the set's midpoint falls outside the region entirely. |
| Compactness | The size of the set: the span from the first oligo's start to the last one's end. Smaller is one diffraction-limited spot. |
| Even spacing | Homogeneity: the inverse standard deviation of the gaps between consecutive oligos. Higher means more evenly spread; clumped oligos leave dark stretches. |
Across the whole design
| Readout | What it is |
|---|---|
| Coverage | The fraction of the target covered by at least one oligo, counted base by base so overlapping oligos are counted once. |
| Gaps | Every stretch the design leaves uncovered, with its length — measured, not eyeballed off the picture. |
| Melting-temperature and G+C distributions | Drawn against the window you asked for, because a set that fills its whole permitted range will not hybridize as one set even though every member passes. |
| Stage accounting | How many windows each stage took in, how many it passed, and the named reason for every rejection — melting temperature too low or too high, G+C outside the window, a prohibited run, an ambiguous base, an off-target site. |
Padlock probes
A padlock probe is one oligo whose two ends, the arms, hybridize side by side on the target so that ligase can close it into a circle, which is then amplified and read by sequencing the barcode it carries. It reports only if the arms ligate, so the question that matters is different from hybridization: whether any other transcript matches across the junction where the two arms meet.
The target is walked in windows twice the arm length. Each window whose melting temperature falls inside the window you set is exported as FASTA; you search those with BLAST against your transcriptome and paste the tabular output back. A window is rejected if any other transcript matches more than half of it at over 80% identity and the match spans the ligation site with five bases to spare on each side. Non-overlapping windows are then packed from the 3′ end, and each is assembled into its padlock: the 3′ arm, the upstream linker, the barcode, the downstream linker and the 5′ arm.
Per padlock target
| Readout | What it is |
|---|---|
| Melting temperature | Nearest-neighbour Tm (Sugimoto 1996 parameters) of the two-arm target at 0.1 µM probe, 75 mM monovalent and 10 mM divalent salt, corrected for 20% formamide. These conditions are fixed, as the published pipeline fixes them. |
| Junction verdict | Whether any other transcript in your BLAST output covers the ligation site at the identity and length above. Transcripts you name as variants of the target (its isoforms) count as the target itself. |
| Found in the database | Whether BLAST found the target itself at full length and 100% identity. A target that was not is dropped from the final list, since nothing confirms it is the transcript you meant. |
| Start and padlock sequence | Where the target begins on the molecule, 1-based, and the full probe sequence to order. |
Across the design: how many windows melt in range, how many pass the junction check, and how many fit without overlapping at the spacing you set.
What is not claimed. The junction check is only as complete as the database you searched: a transcriptome that lacks a paralog cannot reject a probe against it. The backbone defaults are placeholder text, not sequence, and the reading says so until you set real linkers and a barcode. The published pipeline also offers a random subset of N probes per gene; that step is not offered, because a random draw is not a result you could reproduce.
HCR split-initiator probes
HCR v3.0 (hybridization chain reaction) splits each probe into two halves that bind side by side on the target, 25 nt each around a 2 nt gap. Each half carries half of an amplifier initiator, so the fluorescent hairpins polymerize only where both halves have bound; one half bound alone starts nothing, which is what suppresses background without a wash-stringency search.
Every 52 nt window of the target is reverse-complemented into a candidate probe and filtered in a fixed order: a run of C, then of G (seven bases holding at least five of one base), a hairpin that melts at or above 45 °C, more alignments than you allow, G+C outside 45–55% and an RNA
free energy outside −70 to −50 kcal/mol. Optionally the two halves must melt within a few degrees of each other. From what survives, the windows closest to the target free energy are taken one at a time, skipping any that overlaps one already taken, and each is split and given the initiator halves of the amplifier you choose (B1 to B5, one per colour when you multiplex).Uniqueness comes from an alignment you make: export the tiles as FASTA, align them with
bowtie2 -k 3 -f against your genome or transcriptome, and paste the SAM back. Without one, no
uniqueness filter runs and the reading says so.
Per pair
| Readout | What it is |
|---|---|
| Start | Where the 52 nt window begins on the target, 1-based. |
| Free energy (ΔG) | The RNA duplex free energy of the whole probe at 37 °C, from Sugimoto 1995's hybrid nearest-neighbour parameters with an initiation term and a salt correction. Pairs are ranked by how close this is to the target. |
| G+C | Percent G+C over the whole window, the two gap bases included. |
| Half ΔTm | How far apart the two halves' melting temperatures are (primer3's nearest-neighbour Tm at 50 mM Na⁺, 1.5 mM Mg²⁺). Shown on every pair; used as a filter only when you turn the check on. |
| Hits | How many alignments the tile has in your SAM, when you gave one. |
| Odd and even oligo | The two sequences to order: the odd initiator half before the 3′ half-probe, and the 5′ half-probe before the even initiator half. |
Across the design: how many windows each filter kept, in the order they ran, and how many pairs were selected. When fewer pairs come back than you asked for, the reading says the filters left no more non-overlapping windows.
What is not claimed. A tile that spans an exon junction aligns nowhere in genomic sequence, so a genome alignment passes it as unique; that is right for a genome mask and is why the transcriptome is the other choice. The free energy is for the probe as one 52 nt duplex, not for each half.
Controls
Oligos
| Control | Default | What it sets |
|---|---|---|
| Length | — | How long each candidate oligo is |
| Tm | — | The melting-temperature window a candidate must fall in |
| G+C | — | Its G+C window, as a percentage |
| Spacing | 0 nt | Bases forced between consecutive oligos |
| Prohibited runs | AAAAA, TTTTT, CCCCC, GGGGG | Subsequences an oligo may not contain. Homopolymer runs hybridize promiscuously, which is why they are banned by default |
| Allow overlapping oligos | off | Off by default: two oligos that overlap cannot be used together in one probe |
Buffer
| Control | Default | What it sets |
|---|---|---|
| Sodium | 390 mM | The monovalent salt the melting temperature is computed at |
| Probe strand | 25 nM | The higher-concentration strand in that calculation |
| Target strand | 25 nM | The lower-concentration strand. The calculation uses probe − target/2 |
Specificity
| Control | Default | What it sets |
|---|---|---|
| Decision | Trained model | Or Unique alignment only, or No alignment at all |
| Hybridization temperature | 42 °C | The model was trained at six temperatures. Its coefficients change sign across the range, so an intermediate value would be a different model rather than a blend of two |
| Alignment (SAM) | empty | The aligner's own output for these candidates |
Probe set
| Control | Default | What it sets |
|---|---|---|
| Gather oligos into a probe | off | Off returns every surviving oligo. On picks the set that images best: compact, evenly spread and centered on the molecule |
| Oligos per probe | 24 | How many oligos a probe holds, from 2 to 500 |
| Band defined by · Ranked by | Centrality on the region · Compactness | Which property narrows the candidate sets and which orders them. Either can be centrality on the region, compactness or even spacing |
| Band width | 0.1 | How far a set may sit from the best one on the band property, as a fraction of its value |
| Probes returned | 1 | How many probe sets come back |
HCR pairs
| Control | Default | What it sets |
|---|---|---|
| Channel | B1 | The amplifier whose initiator halves the oligos carry: B1 to B5 |
| Max probes | 20 | How many pairs to select at most |
| Min GC · Max GC | 45 · 55 % | The G+C window of the whole window |
| Min Gibbs · Max Gibbs · Target Gibbs | −70 · −50 · −60 kcal/mol | The free-energy window, and the value pairs are ranked toward |
| Tile size | 52 nt | Advanced. The window that becomes one pair |
| Max run length · Max run mismatches | 7 · 2 | Advanced. A C or G run is this many bases holding all but this many of one base |
| Max hairpin Tm | 45 °C | Advanced. A window whose hairpin melts at or above this is dropped |
| ΔTm filter · ΔTm max | off · 5 °C | Advanced. Drop pairs whose halves melt further apart than this |
| Alignment (SAM) | empty | bowtie2's output for the exported tiles |
| Num hits allowed | 1 | Shown once a SAM is pasted. Alignments a tile may have and still pass |
Uncertainty, and what is not claimed
The specificity screen is only as good as the alignment you give it. If you do not supply one, the oligos are returned unscreened and the reading says so in those words — they are candidates, not probes. Nothing here infers specificity from the sequence alone.
The off-target model is a classifier, not a measurement. It returns a probability, and you choose the threshold. It was trained at six hybridization temperatures and its coefficients change sign across that range, so a temperature it was not trained at is refused rather than interpolated: a blend of two models is not a model.
Melting temperatures are predictions. They are nearest-neighbour estimates under the conditions you state, and the formamide correction in particular is a linear approximation of roughly 0.65 °C per percent.
Provenance of correctness
The three stages are checked against the published tools that define them: the tiling and the specificity model against OligoMiner's own programs, and the probe-set selection against the iFISH probe-design package. The padlock design is checked against the Nilsson lab's own padlock pipeline, on real BLAST output for every RefSeq transcript of a human chromosome region. The HCR pairs are checked against the Goff lab's HCRProbeDesign over the same transcripts at four settings, with real bowtie2 alignments, and the initiator halves against Choi et al. 2018. In each case the check regenerates the output those authors themselves published, and compares it exactly rather than approximately.
Practical notes
- Formamide moves everything. A 50% formamide buffer lowers a 78 °C oligo to about 46 °C, so the melting-temperature window you set is meaningless without it. It is a control here, not a constant.
- Widen the length range before you widen the temperature window. The tiler grows a window from the shortest permitted length until one fits, so a 36–41 nt range finds probes a fixed 36 nt cannot.
- Look at the gaps, not just the count. Forty oligos that all sit in the first third of a transcript is a worse probe than twenty spread across it, and only the coverage track shows that.
- Click an oligo on the track to select those bases on the sequence map. Designed oligos and padlock targets and HCR pairs are also drawn on the map itself, and disappear when the panel closes.
- Name the isoforms. Two isoforms share most of their exons, so without naming them as variants of the target, almost every padlock window reads as off-target against its sibling.