Spatial and cellular mapping of biomolecules in situ by high-throughput sequencing
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.
1. A method for single cell mapping comprising
(a) labeling template nucleic acid sequences in fixed cells with unique molecular identifiers (UID's), wherein the labeling comprises adding at least one α-UID to each template nucleic acid, thereby generating uniquely labeled template nucleic acids;
(b) amplifying the labeled template nucleic acids using in situ amplification the amplification reaction also further concatenating individual amplified labeled template nucleic acids together such that each concatenation event results in incorporation of at least one unique β-UID, wherein the frequency of concatenation events between amplified labeled template nucleic acids is a function of a proximity of each individual labeled template nucleic acid to another labeled template nucleic acid; and
(c) generating a spatially resolved physical mapping of each cell of the fixed cells by isolation and sequencing of the uniquely labeled concatenation events from (b).
2. The method of claim 1 , wherein the nucleic acid is DNA.
3. The method of claim 2 , wherein the DNA is complementary DNA (cDNA).
4. The method of claim 1 , wherein the nucleic acid is RNA.
5. The method of claim 1 , wherein the cell is fixed and permeabilized.
6. The method of claim 1 , wherein the at least one UID is a completely random or computationally designed 10 to 50 mer between a gene-specific and adapter region of a primer used for reverse transcription.
7. The method of claim 1 , further comprising cross-linking amplification products of steps (b) and/or (c).
8. The method of claim 7 , wherein the cross-linking further comprises an additional UID-labeling step.
9. The method of claim 7 , wherein the cross-linking comprises overlap extension polymerase chain reaction.
10. The method of claim 7 , wherein the cross-linking comprises associating multiple amplification products with ligase or capture beads.
11. The method of claim 1 , wherein the amplification comprises thermocycling or isothermal amplification.
12. The method of claim 1 , wherein the amplification occurs in solution, in a hydrogel, or on a solid support.