Compositions and methods for gene expression library analysis

A method of gene expression analysis, comprising:

• a) contacting a first RNA analyte and a second RNA analyte in a cell or tissue sample with a first probe and a second probe, respectively, to generate a first amplicon and a second amplicon of a library of amplicons,
• wherein the first probe is a first padlock probe comprising a 3′hybridization region and a 5′ hybridization region complementary to the first RNA analyte in the arms of the first padlock probe and the second probe is a second padlock probe comprising a 3′hybridization region and a 5′ hybridization region complementary to the second RNA analyte in the arms of the second padlock probe,
• wherein the first amplicon comprises i) a first sequencing priming site and ii) a first identifier sequence, and the second amplicon comprising i) a second sequencing priming site different from the first sequencing priming site and ii) a second identifier sequence,
• wherein the first and second identifier sequences comprise a sequence derived from the first RNA analyte and the second RNA analyte, respectively;
• b) using a first sequencing primer to sequence the first identifier sequence of the first amplicon,
• c) removing, cleaving, or blocking the extension product of the first sequencing primer;
• and
• d) using a second sequencing primer different from the first sequencing primer to sequence the second identifier sequence of the second amplicon,
• thereby using the sequenced first and second identifier sequences to identify the first RNA analyte and the second RNA analyte.