MAPseq and BARseq

High-throughput mapping of single neuron projections by sequencing of barcoded RNA

"Here we describe MAPseq (Multiplexed Analysis of Projections by Sequencing), a technique that can map the projections of thousands or even millions of single neurons by labeling large sets of neurons with random RNA sequences ("barcodes"). Axons are filled with barcode mRNA, each putative projection area is dissected, and the barcode mRNA is extracted and sequenced. Applying MAPseq to the locus coeruleus (LC), we find that individual LC neurons have preferred cortical targets. By recasting neuroanatomy, which is traditionally viewed as a problem of microscopy, as a problem of sequencing, MAPseq harnesses advances in sequencing technology to permit high-throughput interrogation of brain circuits."

In the Rosetta brains paper, they used "fluorescent in-situ sequencing of barcoded individual neuronal connections (FISSEQ-BOINC)". See also in situ sequencing.

RNA barcoding paper ("multiplexed analysis of projections by sequencing")

"yeah that is probably the coolest thing ever" - 2016

"reducing this to a sequencing problem is an excellent idea because we have become very good at dna sequencing"

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

Other

Network cloning using DNA barcodes (2016)

MAPseq and BARseq core facility

MAPseq-uencing long-range neuronal projections

Spatial organization of projection neurons in the mouse auditory cortex identified by in situ barcode sequencing (2018) and https://x.com/TonyZador/status/981693522276487174

Reconstruction of 1,000 projection neurons reveals new cell types and organization of long-range connectivity in the mouse brain (2019)

BAcTrace a new tool for retrograde tracing of neuronal circuits (2020)

SYNseq

Using high-throughput barcode sequencing to efficiently map connectomes (2017)

"Here we present SYNseq, a method for converting the connectome into a form that can exploit the speed and low cost of modern high-throughput DNA sequencing. In SYNseq, each neuron is labeled with a unique random nucleotide sequence--an RNA "barcode"--which is targeted to the synapse using engineered proteins. Barcodes in pre- and postsynaptic neurons are then associated through protein-protein crosslinking across the synapse, extracted from the tissue, and then joined into a form suitable for sequencing. Although at present the inefficiency in our hands of barcode joining precludes the widespread application of this approach, we expect that with further development SYNseq will enable tracing of complex circuits at high speed and low cost."

"To translate anatomical questions to a format amenable to sequencing, we label neurons uniquely with random nucleic acid sequences ("barcodes"). As a first proof of principle, we recently described MAPseq, a method for reading out long range projections with single neuron resolution [18]. In MAPseq, we infect neurons with a pool of barcoded virus particles and thus uniquely label e very infected neuron with the barcode sequence carried by the viral particle that infected the neuron. The barcode is then expressed as an mRNA and is transported into axons, where we detect the barcode mRNA by sequencing as a proxy for the axonal projection of every labeled neuron. MAPseq allows the simultaneous tracing of thousands and potentially millions of single neuron projections - presenting a speedup of up to five orders of magnitude over traditional, microscopy-based methods. While MAPseq provides information about area - to - area connectivity at single neuron resolution, it does not provide single-neuron information about neuron-to-neuron connectivity."

"Here, we introduce SYNseq, a method for converting synaptic connections into a form suitable for readout by high-throughput DNA sequencing. SYNseq consists of four steps: neuronal barcoding, trafficking of barcodes to the synapses via tight association with engineered synaptic proteins, joining of barcodes into a form suitable for sequencing, and reconstruction of the network connectivity (Fi g. 1). Briefly, a pre-synaptic mRNA barcode is trafficked to the presynaptic terminal via association with an engineered version of the Neurexin1B (Nrx1B) protein. Likewise, the postsynaptic barcode is trafficked to the postsynaptic terminal via association with a modified Neuroligin1AB (Nlg1AB) protein. Across a synapse, the presynaptic SYNseq components are covalently linked to the postsynaptic SYNseq components and then immunoprecipitated for further biochemical manipulation to link the pre- and postsynaptic barcodes."

Connectome-seq

Connectome-seq: high-throughput mapping of neuronal connectivity at single-synapse resolution via barcode sequencing (2025)

"Connectome-seq relies on the labeling of synaptic connections using engineered protein-RNA complexes to translate a synapse into a pair of unique RNA barcodes. The intended outcome is that one barcode will be associated with the pre-synaptic neuron and the other with the post-synaptic neuron, allowing for later profiling via high-throughput sequencing."

"Two protein anchors, termed SynBar (synaptic barcoding), were designed: PreSynBar to target presynaptic terminals and PostSynBar to target postsynaptic sites. PreSynBar consists of a modified neurexin 1β protein fused to the large fragment of split-GFP (GFP1-10) and an RNA-binding domain, λN22 peptide, similar to other reported designs. PostSynBar is based on neuroligin 1, also fused to the complementary small fragment of split-GFP (GFP11) and the same RNA-binding domain."

"These trans-membrane protein constructs are designed to interact at synapses, reconstituting GFP and bringing together their associated RNA barcodes to anchor at the synaptic membranes. The protein scaffolds were largely adopted from the SynView constructs, where split-GFP fragments are embedded inside the synaptic proteins rather than at their N-terminals."

"Nuclei contain both cellular transcriptomes and copies of either PreRNA or PostRNA, allowing identification of cell types and their associated barcodes. During gentle brain tissue homogenization, the cell membranes connecting pre-synaptic terminals to axons and post-synaptic sites to dendrites are physically separated. The exposed membrane surfaces at both the pre- and post-synaptic compartments then reseal, forming structures called synaptosomes. Since PreSynBar and PostSynBar proteins are connected across the synaptic cleft, tightly bound to the associated RNA barcodes, these protein-RNA complexes should remain intact as long as the synaptic interface is preserved. This allows synaptosomes to maintain the paired PreRNA and PostRNA information from connected neurons, providing direct evidence of synaptic connections. By matching barcodes between nuclei and synaptosomes, we can link the identity of connected neurons and map their connectivity patterns."

"Workflow for parallel isolation of nuclei and synaptosomes. Following AAV injection into pre- and postsynaptic regions, brain tissue is processed through differential centrifugation. Slow spins yield crude pre- and post-nuclei, while a fast spin of the supernatant of the slow spin produces the synaptosome fraction."

processed about 20,000 synaptosomes/hour

See also Network cloning using DNA barcodes (Zador 2016/2019) and https://gnusha.org/logs/2025-09-30.log for an idea to reconstitute the biological topology from genomic data. As mentioned on brain uploading.

PRISM

Combinatorial protein barcodes enable self-correcting neuron tracing with nanoscale molecular context and E11-prism

"E11 Bio’s approach bridges the gap with protein barcodes: abundant, cell-filling tags that permit transit-style neuron tracing at the scale of molecular barcodes. In our paper, we estimate that our PRISM technology creates barcode diversity >750-fold greater than previous multicolor approaches while retaining strong cell-filling labelling."

"We also show how PRISM can go beyond neuron tracing by annotating neurons with direct molecular measurements such as synapse composition"

"Protein barcoding: At the heart of PRISM is the concept of cellular barcodes. Neurons are engineered to express a random subset of antigenically-distinct, cell-filling proteins (‘protein bits’). Thanks to the power of combinatorics, the palette of potential unique barcodes rapidly expands as the number of available proteins increases. With fluorescent proteins, there are ~27 = 128 possible binary combinations. In our paper, we demonstrate 18 bits (218 − 1 = 262,143 possible binary combinations)."

"The repeated rounds of immunostaining allow many different targets to be visualized in each cell. In our paper we image 23 targets: 18 protein bits and 5 molecular markers."

"The key to our approach is a library of designed “protein bits”. Each protein variant represents a bit in a barcode, and are based on GFP proteins fused to short, antigenically distinct peptide tags. We designed 18 variants in total. We used a simple C-terminal fusion structure with minimal modification of the GFP, thus maintaining important characteristics of the base protein (i.e. cell-filling labelling). We created a pool of viruses (AAV), where each virus encodes one of these protein bits. When the pool is injected into the mouse brain, multiple viruses stochastically infect each neuron. Each cell thus expresses a subset of bits to create a protein barcode."