US 12,270,074 B1
Compositions and methods for gene expression library analysis
Patrick J. Marks, San Francisco, CA (US); and Michael Schnall-Levin, San Francisco, CA (US)
Assigned to 10X GENOMICS, INC., Pleasanton, CA (US)
Filed by 10x Genomics, Inc., Pleasanton, CA (US)
Filed on Feb. 27, 2024, as Appl. No. 18/589,354.
Application 18/589,354 is a continuation of application No. PCT/US2023/021925, filed on May 11, 2023.
Claims priority of provisional application 63/340,730, filed on May 11, 2022.
Int. Cl. C12Q 1/6869 (2018.01); C12N 15/10 (2006.01); C12Q 1/48 (2006.01); C12Q 1/6874 (2018.01)
CPC C12Q 1/6869 (2013.01) [C12N 15/1086 (2013.01); C12Q 1/485 (2013.01); C12Q 1/6874 (2013.01)] 20 Claims
 
1. A method of gene expression analysis, comprising:
a) providing a library of amplicons within a cell or tissue sample attached to a solid support-by chemical bonding, physical forces or a coating on the solid support,
wherein the library of amplicons comprises a first amplicon comprising i) a first sequencing priming site and ii) a first identifier sequence, and a 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 of a first analyte and a second analyte, respectively, in the cell or tissue sample;
b) using a first sequencing primer to sequence the first identifier sequence of the first amplicon,
wherein the sequencing comprises using a polymerase to incorporate a plurality of cognate nucleotides into the first sequencing primer or an extension product thereof;
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,
wherein the sequencing comprises using a polymerase to incorporate an additional plurality of cognate nucleotides into the second sequencing primer or an extension product thereof;
wherein the plurality of cognate nucleotides and additional plurality of cognate nucleotides in b) and d), respectively, have the structure:

OG Complex Work Unit Chemistry
wherein B is selected from the group consisting of a purine base, a pyrimidine base, and a derivative of a purine or pyrimidine base, and B is coupled to a label via a linker; R1 is a terminator moiety; and R2 is H;
thereby using the sequenced first and second identifier sequences to identify an expressed transcript associated with a member of the library of amplicons.