	US 12,188,087 B2
	Sequential decoding of nucleic acids
Malte Kühnemund, Stockholm (SE); and Toon Verheyen, Solna (SE)
Assigned to 10x Genomics, Inc., Pleasanton, CA (US)
Filed by 10x Genomics, Inc., Pleasanton, CA (US)
Filed on Nov. 29, 2021, as Appl. No. 17/537,306.
Application 17/537,306 is a continuation of application No. PCT/EP2020/065090, filed on May 29, 2020.
Claims priority of application No. 1907752 (GB), filed on May 31, 2019; application No. 1907764 (GB), filed on May 31, 2019; application No. 1907772 (GB), filed on May 31, 2019; and application No. 1907779 (GB), filed on May 31, 2019.
Prior Publication US 2022/0213529 A1, Jul. 7, 2022
Int. Cl. C12P 19/34 (2006.01); C12Q 1/6813 (2018.01); C12Q 1/6816 (2018.01); C12Q 1/6874 (2018.01); C12Q 1/6844 (2018.01)

CPC C12Q 1/6874 (2013.01) [C12Q 1/6813 (2013.01); C12Q 1/6816 (2013.01); C12Q 1/6844 (2013.01); C12Q 2537/1373 (2013.01)]

17 Claims

1. A method of analyzing a sample comprising a target nucleic acid analyte, comprising:

a) contacting the sample with a probe that binds to the nucleic acid analyte,

wherein the probe comprises a barcode sequence that identifies the nucleic acid analyte,

wherein the barcode sequence is associated with a signal code sequence comprising a sequence of optical signals that identify the barcode sequence, and

wherein the sample is a permeabilized cell or tissue sample on a solid substrate;

b) circularizing the probe;

c) amplifying the circularized probe to produce a rolling circle amplification (RCA) product in the sample, the RCA product comprising multiple complementary copies of the barcode sequence;

d) contacting the sample with a first detection probe and a first reporter probe to generate a first complex comprising the first detection probe hybridized to the RCA product and the first reporter probe hybridized to the first detection probe,

wherein the first detection probe comprises (i) a recognition sequence that binds to a complementary copy of the barcode sequence and (ii) a first overhang sequence, and

wherein the first reporter probe comprises (i) a sequence complementary to the first overhang sequence and (ii) a first optically detectable moiety;

e) imaging the sample to detect the first optically detectable moiety, thereby detecting a first optical signal of the signal code sequence;

f) removing the first detection probe from the RCA product;

g) contacting the sample with a second detection probe and a second reporter probe to generate a second complex comprising the second detection probe hybridized to the RCA product and the second reporter probe hybridized to the second detection probe,

wherein the second detection probe comprises (i) a recognition sequence that binds to a complementary copy of the barcode sequence and (ii) a second overhang sequence, and

wherein the second reporter probe comprises (i) a sequence complementary to the second overhang sequence and (ii) a second optically detectable moiety;

h) imaging the sample to detect the second optically detectable moiety, thereby detecting a second optical signal of the signal code sequence,

wherein the signal code sequence comprising at least the first optical signal and the second optical signal is detected at a location in the sample; and

i) using the signal code sequence to identify the nucleic acid analyte at the location in the sample.