Spatially distinguished, multiplex nucleic acid analysis of biological specimens

A method for spatially tagging target nucleic acids of a biological specimen, comprising:

• (a) providing a plurality of nucleic acid primers attached to a solid support, wherein the nucleic acid primers in the plurality comprise a universal primer sequence that is common to the nucleic acid primers in the plurality;
• (b) randomly distributing and binding a population of nucleic acid probes to the plurality of nucleic acid primers, wherein the nucleic acid probes comprise a universal primer binding sequence that hybridizes to the universal primer sequence, a target capture sequence, and a spatial tag sequence that differs from spatial tag sequences of other nucleic acid probes in the population, thereby attaching the nucleic acid probes at randomly located positions on the solid support;
• (c) amplifying the nucleic acid probes by extension of the nucleic acid primers, thereby producing nucleic acid clusters having copies of the spatial tag sequence and target capture sequence at the randomly located positions on the solid support;
• (d) performing a nucleic acid detection reaction to determine the spatial tag sequences at the randomly located positions on the solid support, thereby determining the position of each of the spatial tag sequences on the solid support;
• (e) contacting a biological specimen with the nucleic acid clusters on the solid support;
• (f) hybridizing the target capture sequences of the nucleic acid clusters to target nucleic acids from portions of the biological specimen that are proximal to the nucleic acid clusters;
• and
• (g) extending the target capture sequences to produce extended probes that comprise sequences of the target nucleic acids, or portions thereof, and the copies of the spatial tag sequences, thereby spatially tagging the target nucleic acids of the biological specimen.