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

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 postsynap tic SYNseq components and then immunoprecipitated for further biochemical manipulation to link the pre- and postsynaptic barcodes."