In Situ Sequencing (ISS)

ISS workflows bifurcate into two distinct strategies:

• Targeted ISS: Uses padlock probes designed against a pre-selected panel of genes (typically 100-300). This provides higher sensitivity for known transcripts and is the standard for applications like Cartana (10x Genomics) and HybISS.
• Untargeted ISS: Captures the entire polyadenylated transcriptome using random primers, enabling true discovery without prior gene selection. This approach, exemplified by FISSEQ, trades sensitivity for breadth.

The foundational signal amplification step that makes single-molecule detection possible:

• A circular DNA template is localized to the transcript.
• A phi29 DNA polymerase generates a long, tandem-repeat concatemer (RCP) covalently anchored to the target site.
• This creates a diffraction-limited fluorescent spot containing hundreds to thousands of identical sequence copies, boosting the signal-to-noise ratio sufficiently for microscopy-based detection.

ISS reads nucleotide identity through iterative ligation rather than polymerase extension:

• Fluorescently labeled, degenerate nonamer probes are hybridized and ligated to an anchor primer.
• Each probe queries a single base position with a specific fluorophore.
• After imaging, the fluorophore and a portion of the probe are cleaved, resetting the strand for the next cycle.
• This chemistry avoids homopolymer errors common to sequencing-by-synthesis and is optimized for short reads (4-6 bases) sufficient for gene identification.

Gene identity is decoded from short, gene-specific barcode sequences rather than full transcript reconstruction:

• A 4-base barcode sequence can theoretically distinguish 256 genes; 6 bases can resolve over 4,000.
• Error-correcting barcodes (e.g., Hamming distance codes) are employed to mitigate single-base sequencing errors.
• The barcode readout is matched against a reference library to assign each fluorescent spot to a specific gene, enabling simultaneous identification of hundreds of transcripts in a single cycle.

ISS achieves true single-molecule resolution, localizing transcripts to their precise subcellular coordinates:

• Individual RCPs are resolved as discrete, punctate spots using high-NA objectives.
• This enables analysis of RNA localization to specific organelles, dendrites, or cellular compartments.
• Unlike spot-based capture methods (e.g., Visium), ISS can distinguish transcripts in adjacent cells and even within different regions of the same cell, revealing spatial dynamics of splicing and trafficking.

The sequential imaging chemistry enables high multiplexing within a single tissue section:

• Each sequencing cycle images 4 channels (one per base: A, C, G, T).
• After imaging, a chemical stripping or photocleavage step removes the fluorophores.
• This cycle repeats for each base position in the barcode.
• The result is a multi-channel, multi-cycle image stack where each spot's color sequence across cycles reveals its gene identity, enabling hundreds of genes to be mapped simultaneously.