MAPseq & BARseq

Type: Molecular / Cellular,

Keywords: Molecular Barcode, Cellular Barcoding and Sequencing, Gene Expression, Mapping Method, Next-Generation Sequencing, Single-Cell, High Throughput, Long-Range Projections

Resource ID: N/A

Mapping long-range projections at single neuron resolution using BARseq and MAPseq

BARseq and MAPseq can map long-range projections of 1000s of neurons in a single brain area at single neuron resolution, and further correlate projections to gene expression and Cre. We achieve mapping of densely labeled neurons at single neuron resolution by cellular barcoding and sequencing. Our methods allow comparison of projections across neuronal subtypes within an animal, across individual animals, and across genotypes. We offer MAPseq service through CSHL core facility, and may offer BARseq service in the future. We also welcome other labs to adopt both methods on their own.

* MAPseq, a multiplexed barcode-assisted neuronal projection mapping method using next-generation sequencing.
* A technique used to map the connections of different brain cells and gain a better understanding of how they interact with each other.
* Developed by Zador and his collaborators, uses genetic barcodes to label cells, making it possible to map hundreds or potentially thousands of individual neurons within this tangle.
* MAPseq labels each neuron with a unique molecular barcode that travels down the axon, marking the entire cell. Researchers can determine where a neuron projects by dissecting brain regions of interest and searching for the barcode.
* BARseq, the next generation of MAPseq.
* A method for efficient and accurate sequencing of cellular barcodes in situ.
* BARseq, in which barcodes are sequenced in intact brain slices. This approach preserves anatomical information and allows scientists to examine connectivity at greater resolution.
* Improved version of the gap padlock probe-based method with a five-fold increase in efficiency.
* BARseq allows researchers to map multiple measures in the same neurons: the bar codes, neuron activity via calcium imaging and gene-expression data.
* This new technology can be used to expand the brain map by accurately pinpointing the location of a neuron.
* Determines a neuron’s connections, its pattern of gene expression and its physiological activity.
* A valuable tool for studying how neural circuits are formed. *Five-fold increase in amplification efficiency, with a sequencing accuracy of at least 97%.
* Compatible with Illumina sequencing by synthesis (SBS), which has a higher signal-to-noise ratio.
* Both use labels based on RNA rather than fluorescent markers or dyes to trace neuronal projections.

* Mapped the connections of 3,579 neurons in the auditory cortex of the mouse brain.
* Demonstrated the accuracy and efficiency by sequencing random barcodes expressed in cells in culture.
* Used this approach to efficiently trace hundreds of cells in the visual cortex.
* Determined the projection patterns of 591 individual neurons in the mouse primary visual cortex using whole-brain fluorescence-based axonal tracing and high-throughput DNA sequencing of genetically barcoded neurons (MAPseq).
* Identified gene correlates of projections in a subpopulation of cortical neurons in both auditory and motor cortex using BARseq2

* Capable of mapping thousands of neurons in a single mouse, at single neuron resolution.
* Could be used for barcode-assisted lineage tracing, and to map long-range neuronal projections.
* Used the techniques to identify different types of neurons in the auditory cortex.
* First confirmed the technique works, using MAPSeq data to identify known types of projection neurons. Then used BARseq to further subdivide these categories and examine whether cells that project to similar brain areas reside in similar laminar layers.

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* Less expensive.
* Less labor-intensive.
* Less time-consuming.
* BARseq allows researchers to tag and sequence the neurons in situ, or in their original form and location on the brain.
* Can pinpoint exactly where a neural connection begins.
* In situ bar-code sequencing would potentially allow visualization of the morphology of individual neurons.
* Preserving the locations of the cells being sequenced would allow correlation of lineages and projections with other information, such as gene expression assayed through FISH or in situ sequencing, and neuronal activities assayed through functional imaging, at cellular resolution.
* The approach is efficient, labeling thousands of neurons in a single experiment.
* BARseq employs the same sequencing reaction and reagents as Illumina sequencing machines, a popular commercial sequencing technology. But the reaction is done in a brain slice under a microscope.

* MAPSeq requires scientists to remove and analyze segments of brain tissue, destroying fine scale detail.
* BARseq can only provide a close estimate of where a neural connection ends.
* Do not have enough spatial resolution at the destination.

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* Kebschull et al 2016 High-Throughput Mapping of Single-Neuron Projections by Sequencing of Barcoded RNA. Neuron 91(5)975-987. doi: 10.1016/j.neuron.2016.07.036

*Han et al. 2018, The logic of single-cell projections from visual cortex, Nature 556: 51–56.

* Chen et al. 2019, High-throughput mapping of long-range neuronal projection using in situ sequencing. Cell. 179(3), 772-786. doi: 10.1016/j.cell.2019.09.023.

* Sun et al. 2020, Integrating barcoded neuroanatomy with spatial transcriptional profiling reveals cadherin correlates of projections shared across the cortex. bioRxiv 2020.08.25.266460; doi: 10.1101/2020.08.25.266460.

http://zadorlab.labsites.cshl.edu/publications-3/

* Kebschull et al 2016 High-Throughput Mapping of Single-Neuron Projections by Sequencing of Barcoded RNA. Neuron 91(5)975-987. doi: 10.1016/j.neuron.2016.07.036

* Han et al. 2018, The logic of single-cell projections from visual cortex, Nature 556: 51–56.

* Chen et al. 2019, High-throughput mapping of long-range neuronal projection using in situ sequencing. Cell. 179(3), 772-786. doi: 10.1016/j.cell.2019.09.023.

* Sun et al. 2020, Integrating barcoded neuroanatomy with spatial transcriptional profiling reveals cadherin correlates of projections shared across the cortex. bioRxiv 2020.08.25.266460; doi: 10.1101/2020.08.25.266460.

https://www.nature.com/articles/nature26159

https://pubmed.ncbi.nlm.nih.gov/?term=mapseq+zador

https://pubmed.ncbi.nlm.nih.gov/27545715/

https://pubmed.ncbi.nlm.nih.gov/31626774/

https://www.biorxiv.org/content/10.1101/2020.08.25.266460v1

 

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CONTACT NAME, POSITION

Xiaoyin Chen (Postdoc)
Anthony Zador (Professor)

ORGANIZATION

CSHL

CONTACT INFORMATION

TEAM

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LAB WEBSITE(S)

GRANT SUPPORT / FUNDING SOURCE

* National Institutes of Health [5RO1NS073129 to Anthony M. Zador, 5RO1DA036913 to A.M.Z., 5U19MH114821 to A.M.Z.]
* Brain Research Foundation [BRF-SIA-2014-03 to A.M.Z.]
* IARPA MICrONS [D16PC0008 to A.M.Z.]
* Simons Foundation [382793/SIMONS to A.M.Z.]
* Paul Allen Distinguished Investigator Award [to A.M.Z.]
* postdoctoral fellowship from the Simons Foundation to X.C.