In situ sequencing

Reviews

“Museum of spatial transcriptomics” (Nat Methods, 2022) — panoramic field map spanning ISS, MERFISH, seqFISH, analysis trends. Nature
Padlock probe–based in situ sequencing (Annu Rev Genom Hum Genet, 2023) — clear review of targeted ISS chemistry (padlock + RCA), performance, and applications. PubMed, Annual Reviews
Multiplexed spatial transcriptomics methods & the road to multi-omics (Frontiers Cell Dev Biol, 2024) — compares ISS, FISH, NGS-based approaches; discusses ExM combos. Frontiers
Spatial analysis toolkits for RNA ISS (WIREs RNA, 2024) — survey of pipelines for decoding and analyzing ISS data. Wiley Online Library
Spatial transcriptomics in pathology/medicine (Am J Pathol, 2025) — methods + analysis focus with clinical lens. American Journal of Pathology

In situ sequencing (ISS) — key methods

Original ISS in preserved tissue (Nat Methods, 2013) — padlock probes + RCA; targeted detection in FFPE sections. PubMed
FISSEQ protocol (Nat Protocols, 2015) — sequencing-by-synthesis of cDNA amplicons in fixed cells/tissue; genome-wide flavor of ISS. Nature, PMC
HybISS (Nucleic Acids Res, 2020) — sequencing-by-hybridization readout; simpler implementation for targeted panels. Oxford Academic, PubMed
BaristaSeq (Nucleic Acids Res, 2018) — optimized padlock-based in situ barcode sequencing; used for neuronal projection mapping (BARseq). Oxford Academic, PubMed, PMC
STARmap (Science, 2018) — hydrogel-tissue chemistry + in situ DNA sequencing for intact 3D tissue. Follow-on STARmap PLUS adds protein co-detection (Nature, 2023). Science, PMC, PubMed
ExSeq (Science, 2021) — expansion microscopy + in situ sequencing gives nanoscale-resolved RNA maps. Science, PMC
Rapid/crowdedness-robust ISS (bioRxiv, 2022) — faster, higher-density decoding in padlock-based ISS. BioRxiv
Open-source high-throughput targeted ISS (Nat Commun, 2024) — practical, sensitive targeted ISS workflow. PMC

Multiplexed FISH — modern & next-gen

MERFISH (Science, 2015) — error-robust barcoding enables thousands of RNAs per cell; (Vizgen MERSCOPE). Science, PubMed
seqFISH+ (Nature, 2019) — transcriptome-scale (10k genes) imaging with sub-diffraction resolution. PubMed, PMC
osmFISH (Nat Methods, 2018) — cyclic smFISH without barcodes; large-area tissue mapping. PubMed, Nature
HCR RNA-FISH v3.0 (Choi et al., 2018; protocols & 10-plex 2024) — enzyme-free amplification with strong background suppression. PMC, Molecular Technologies, The Journal of Experimental Biology
ClampFISH 2.0 (2022) — fast, scalable amplified RNA FISH; multiplexing. PMC, BioRxiv
π-FISH “rainbow” (Nat Commun, 2023) — multiplex detection of RNA/DNA/proteins in the same tissue. Nature
MERFISH with branched-DNA amplification (2019) — boosts signal, detection efficiency. Nature

Commercial “next-gen” in situ platforms

10x Genomics Xenium — subcellular targeted RNA + optional protein. Nature, PubMed, eLife
NanoString/Bruker CosMx SMI — high-plex RNA(+protein) imaging at single-cell/subcellular scale; several comparative/preprint studies. BioRxiv
Vizgen MERSCOPE (MERFISH) — technical concordance vs RNA-seq and multi-omic co-staining examples. Life Science Alliance, Vizgen

Live-cell “in situ” RNA/DNA imaging (not sequencing)

If you need living tissue/cells, current sequencing methods still require fixation; live-cell work uses CRISPR imaging instead:

LiveFISH / CRISPR–Casilio & related — label genomic loci in live cells, including non-repetitive sites; tracks dynamics. Nature, PubMed
CRISPR-Cas13/Csm RNA imaging — live-cell tracking of endogenous RNAs at single-molecule resolution (reviews + 2025 Nat Biotechnol). PMC, Frontiers, Nature

Software & pipelines

starfish (JOSS, 2021) — open pipeline for image-based transcriptomics (MERFISH/seqFISH/ISS). Journal of Open Source Software, The Open Journal
FISH-quant v2 / Big-FISH (Genome Res, 2022) — modular smFISH detection/segmentation toolkit. PMC, GitHub
PIPEFISH (Patterns, 2023) — CWL-based unified pipeline for FISH spatial transcriptomics. ScienceDirect
Sopa (Nat Commun, 2024) — technology-agnostic, memory-efficient pipeline for image-based spatial omics. Nature

Main methods

Fixed tissue, targeted panels (FFPE or fresh-frozen): HybISS, STARmap/STARmap PLUS, Xenium, CosMx, MERFISH. (HybISS/STARmap for open protocols; Xenium/CosMx/MERSCOPE for turnkey). Oxford Academic, PMC, 10x Genomics, Life Science Alliance
Large gene counts without sequencing chemistry: seqFISH+ (10k genes) if you can handle long imaging runs. PMC
Ultrahigh spatial resolution / nanoscale context: ExSeq (ExM + ISS). Science
Live cells/tissue: CRISPR imaging (DNA with LiveFISH; RNA with Cas13/Csm). (Sequencing in situ still generally requires fixation.) PubMed, PMC

FISSEQ

Core FISSEQ & reviews

FISSEQ, first report (Science, 2014) — untargeted in situ RNA sequencing via cross-linked cDNA “rolonies” and SOLiD SBL imaging. PubMed, Mali
FISSEQ protocol (Nat Protocols, 2015) — detailed, bench-ready workflow; sample prep, crosslinking, imaging cycles, analysis. PMC, Kalhor Lab
Padlock-probe ISS review (Annu Rev Genom Hum Genet, 2023) — complements FISSEQ by covering targeted ISS; good for contrasts and hybrid designs. PubMed
Recent overviews (2023–2025) — succinct sections on FISSEQ vs. other spatial methods and where it fits today. Wiley Online Library, PMC, BioMed Central

“FISSEQ-adjacent” and next-gen alternatives

These all keep the “sequence DNA amplicons in place” idea but change chemistry/physics to boost accuracy, depth, or tissue thickness.

Optics/chemistry to beat crowding & errors

STARmap (Science, 2018) — hydrogel-embedded amplicons + SEDAL two-base ligation decoding for low error in thick tissue (3D). Science, PMC
STARmap PLUS (Nature, 2023) — STARmap with simultaneous protein co-detection; thousands of genes at subcellular voxels. PMC, Nature
ExSeq (Science, 2021) — expansion microscopy + in situ sequencing; comes in untargeted and targeted flavors, enabling long reads and nanoscale mapping. Science, PMC

New ligation/sequencing chemistries

INSTA-seq / PRICKLi (preprint) — bidirectional ligation (paired-end in one cycle) to cut imaging cycles; links in situ UMIs to NGS for longer reads and RNA-protein accessibility info. BioRxiv

Targeted barcode & panel readouts inspired by in situ SBS/SBL

BaristaSeq (NAR, 2018) — optimized padlock-based in situ barcode sequencing (Illumina-style SBS) with higher efficiency/accuracy. Oxford Academic, PubMed
BARseq (Cell, 2019) and BARseq2 (2021) — uses in situ sequencing to map neuronal projections; BARseq2 adds multiplex gene expression readout in the same cells. Recent atlases extend to brain-wide maps. ScienceDirect, PMC, Nature

FISSEQ practical stuff

Hands-on FISSEQ protocol (step-by-step, including FFPE compatibility and imaging). PMC, protocols.io
Pipelines for decoding/analysis: starfish supports FISSEQ/ISS-style assays; 2024 ISS toolkits review covers modern upstream→downstream workflows. Journal of Open Source Software, spacetx-starfish.readthedocs.io, The Open Journal, Wiley Online Library

MAPseq and BARseq

Reviews

Barcode-based connectomics (concept + limitations summary, 2024) — short overview contrasting MAPseq, BRICseq, BARseq, and ConnectID, incl. tradeoffs for adding transcriptomes. eLife
From MAPseq to BRICseq and beyond (2021) — narrative review of barcoded projection mapping from the Zador lab lineage. PMC
Neural circuit research with molecular barcodes (2025) — broader survey placing MAPseq/BARseq among barcoded neurotech. ScienceDirect

MAPseq — essentials

MAPseq (Neuron, 2016) — seminal method: infect neurons with a diverse RNA-barcode virus; bulk-sequence dissected target regions to read one-to-many projections at scale. (Open access.) PMC
MAPseq (primary index / PubMed) — canonical reference & metadata. PubMed
MAPseq2 (preprint, 2025) — protocol update reporting \~3–4× higher barcode detection sensitivity and \~10× lower cost vs. original MAPseq. ResearchGate

BARseq — essentials

BARseq (Cell, 2019) — combines MAPseq with in situ sequencing at the source area to keep somatic/spatial context while still bulk-sequencing targets; maps thousands of neurons in one brain. Cell, PMC
BARseq2 (Neuron, 2021) — adds multiplexed gene-expression readout in the same barcoded neurons (projection + transcript markers in situ). PMC, PubMed
Axonal BARseq (Nat Commun, 2023/2024) — sequences barcodes along axons to resolve branch-specific projection patterns at scale. PMC, Nature

Related / next-gen offshoots

BRICseq (Cell, 2020) — “brain-wide individual-animal connectome sequencing”; a MAPseq variant designed for robust region–region connectivity within single animals and for linking connectivity to activity/genes/behavior. PMC
MERGE-seq (eLife, 2024) — retrograde AAV barcodes + scRNA-seq to jointly profile projectome and transcriptome; paper also benchmarks BARseq/MAPseq/ConnectID tradeoffs. PMC
ConnectID (method referenced in MERGE-seq) — MAPseq + scRNA-seq pairing; raises transcriptome recovery but with modest joint-cell yield. (Cited/quantified within MERGE-seq). eLife
Rabies-based barcoded neuroanatomy (eLife, 2024) — barcoded rabies vectors with in situ and single-cell sequencing for retrograde and trans-synaptic labeling; expands barcode tracing to synaptic hops. eLife
Projection-TAGs (Nat Commun, 2025) — kit-like retrograde AAV toolbox where each target gets a unique RNA barcode for multiplexed tracing + multi-omic profiling; conceptually adjacent to MAPseq/MERGE-seq. Nature

BARseq and MAPseq applications

Max throughput across many targets → MAPseq / BRICseq (bulk-NGS readout; highest cell counts; no somatic imaging). PMC
Keep somatic spatial context; add limited gene panels → BARseq / BARseq2 (in situ sequencing at source; BARseq2 adds tens-to-\~100 genes). Cell, PMC
Resolve axonal branching patterns → Axonal BARseq. PMC
Joint projectome + full scRNA-seq transcriptomes → MERGE-seq / ConnectID (lower joint-cell recovery than pure BARseq2 gene panels, but richer transcriptomes). PMC

Intracellular imaging via in situ sequencing

CODEX multiplexed tissue imaging with DNA-conjugated antibodies

"The CODEX technology was licensed by Stanford University to Akoya Biosciences, and the instrumentation and reagents are now commercially available."

"Co-detection by indexing (CODEX) relies on DNA-conjugated antibodies and the cyclic addition and removal of complementary fluorescently labeled DNA probes" isn't that just Exchange-PAINT? but not superresolution microscopy.

"CODEX shares several limitations with other multiplexed imaging techniques. First, antibodies are expensive. Purified antibodies suitable for use with FFPE tissues generally cost $300––600 per 100 μg; antibodies suitable for FF tissues are often less expensive at ~$100 per 100 μg (provides enough for ~100–200 multicycle stains). Sufficient maleimide-modified DNA oligonucleotides for ~20 antibody conjugations cost ~$800 (source: TriLink BioTechnologies). Fluorescently tagged DNA oligonucleotides sufficient for ~100 reaction cycles cost ~$400 (source: Integrated DNA Technologies). In addition, each antibody requires individual conjugation and validation in a unified staining protocol. Certain antibodies or clones are not suitable for multiplexing (e.g., antibodies may have pH requirement for antigen retrieval that are incompatible with the CODEX unified staining protocol)."

Modified aptamers enable quantitative sub-10-nm cellular DNA-PAINT imaging

"SOMAmer reagents represent a unique class of aptamers that contain modified bases employing hydrophobic residues, similar to the amino acid residues abundantly found in antibody epitopes for high-specificity and high-affinity binding of proteins. These base modifications increase the range of protein targets for which high-affinity ligands can be selected."

aptamers seem to be better from a size perspective, but why did this study not do in situ sequencing of barcodes? looks like direct imaging of a fluorescent hybridizing imager sequence per tagged ?aptamer?

why not ?antibody labeling with DNA tag barcode tails and then do in situ sequencing?

Fluorescent in situ sequencing of DNA barcoded antibodies - this is a very cool article, although needs to be switched over to aptamers instead of antibodies.

Church complains that "Although effective, in situ hybridization (ISH) of oligonucleotides conjugated to antibodies only scales linearly with the number of iterative labelling reactions and does not take advantage of the data density of DNA (4ⁿ where N = the number of bases in the oligos). For instance, one 20 nucleotide-long ISH probe encodes 1 bit of information whereas the sequencing of this length encodes 4²⁰ (over 1 trillion) bits. Recently, Goltsev et al have shown a sequencing based approach to read out the location and identity of barcoded antibodies, however the chemistry chosen did not take advantage of high density base space and therefore drastically reduced the sequence diversity that could be investigated."

In situ sequencing

Reviews