SPATIAL AND CELLULAR MAPPING OF BIOMOLECULES IN SITU BY HIGH-THROUGHPUT SEQUENCING

摘要

The present invention relates to molecular microscopy or volumetric imaging by proximal unique molecular identifiers (“UID”) reaction (“VIPUR”) microscopy methods to record the cellular co-localization and/or spatial distributions of arbitrary nucleic acid sequences, or other biomolecules tagged with nucleic sequences. The method involves one or both of two DNA sequence-components such as an α-UID, which may identify the targeted sequences-of-interest themselves and/or spatial beacons relative to which their distances are measured, and a −β-UID, which labels α-UID association events.

主权项

1. A method for recording cellular co-localization and/or spatial distributions of nucleic acid sequences and/or biomolecules tagged with a nucleic acid in non-dissociated cells or fixed tissue comprising
(a) labeling nucleic acid sequences with unique molecular identifiers (UID's),
wherein the labeling comprises adding at least one α-UID to each nucleic acid,wherein at least one α-UID identifies targeted sequences or biomolecules of interest and wherein after extraction and amplification, all reads from the targeted sequence are identified as originating from individually distinguishable sources,wherein at least one α-UID may identify beacon sequences or biomolecules that are widely expressed, represented within the sample, or externally added via nanoparticles and wherein after extraction and amplification, all reads from the beacon sequence or biomolecules are identified as originating from individually distinguishable sources,wherein the labeling is random or drawn from a pool of pre-defined nucleic acid sequences,thereby generating uniquely labeled target or beacon nucleic acid sequences or biomolecules; (b) in situ amplification of the template of nucleic acid sequences using homogenous heating of the entire sample or selective heating using directed infrared light sources to amplify only selected regions of interest; (c) labeling in situ association events of two or more uniquely labeled template amplification products with at least one β-UID,
wherein the labeling is random or drawn from a pool of pre-defined nucleic acid sequences,thereby generating uniquely labeled nucleic acid sequence-records of in situ association events;and (d) isolation and amplification of the uniquely labeled products from (c), thereby recording cellular co-localization and/or spatial distributions of nucleic acid sequences and/or biomolecules tagged with a nucleic acid in a non-dissociated cell.