Granted Patent B2

US 12,747,475 · App. 18/490,390 · Granted Sep 29, 2026

Inventor: Marlon Stoeckius (Stockholm, SE)

C12Q1/6876

Abstract

Claims (33)

1 . A method of determining a location of a target nucleic acid in a biological sample, the method comprising:

(a) contacting the biological sample with an array comprising a feature, wherein the feature comprises an attached first and second probe, wherein:

a 5′ end of the first probe is attached to the feature;

the first probe comprises in a 5′ to a 3′ direction: a spatial barcode and a capture domain which binds to the target nucleic acid;

a 5′ end of the second probe is attached to the feature;

a 3′ end of the second probe is reversibly blocked; and

the second probe comprises a poly(GI) capture domain;

(b) binding the first probe to the target nucleic acid and extending the first probe to add a sequence that is complementary to a portion of the target nucleic acid to generate a first extended probe;

(c) ligating an adapter to the 5′ end of the target nucleic acid bound to the first extended probe;

(d) adding a sequence complementary to the adapter to the 3′ end of the first extended probe;

(e) after (d), adding non-templated cytosines to the 3′ end of the first extended probe to generate a poly(C) sequence, wherein the poly(C) sequence binds to the poly(GI) capture domain of the second probe;

(f) unblocking the 3′ end of the second probe and extending the 3′ end of the second probe to add a sequence comprising a sequence in the target nucleic acid and a sequence that is complementary to the spatial barcode;

(g) cleaving a region of the second probe at a cleavage site that is 5′ to the poly(GI) capture domain, thereby releasing the second probe from the feature; and

(h) determining (i) the sequence of the spatial barcode or a complement thereof, and (ii) all or a part of the sequence of the target nucleic acid, or a complement thereof, and using the sequences of (i) and (ii) to determine the location of the target nucleic acid in the biological sample.

2 . The method of claim 1 , wherein the poly(GI) capture domain comprises a sequence of (GGI)n, wherein n is about 3 to about 20.

3 . The method of claim 1 , wherein step (h) comprises sequencing the sequence of a nucleic acid molecule comprising the spatial barcode or a complement thereof, and all of a part of the sequence of the target nucleic acid, or a complement thereof.

4 . The method of claim 3 , wherein the sequencing is performed using high throughput sequencing.

5 . The method of claim 1 , wherein the capture domain of the first probe comprises a poly(T) sequence.

6 . The method of claim 1 , wherein the first probe further comprises a unique molecular identifier (UMI), wherein the UMI in the first probe is positioned 5′ to the capture domain in the first probe.

7 . The method of claim 6 , wherein the second probe further comprises a unique molecule identifier (UMI), wherein the UMI in the second probe is positioned 5′ to the poly(GI) capture domain in the second probe and 3′ relative to the cleavage site.

8 . The method of claim 7 , wherein the UMI in the first probe and the UMI in the second probe comprise different sequences.

9 . The method of claim 7 , wherein the UMI in the first probe and the UMI in the second probe comprise the same sequence.

10 . The method of claim 7 , wherein the second probe further comprises a spatial barcode, wherein the spatial barcode in the second probe is 3′ relative to the cleavage site in the second probe.

11 . The method of claim 10 , wherein the spatial barcode in the first probe and the spatial barcode in the second probe comprise different sequences.

12 . The method of claim 10 , wherein the spatial barcode in the first probe and the spatial barcode in the second probe comprise the same sequence.

13 . The method of claim 1 , wherein the target nucleic acid is mRNA.

14 . The method of claim 13 , wherein the mRNA encodes a T-cell receptor or a B-cell receptor.

15 . The method of claim 13 , wherein the mRNA encodes an immunoglobulin heavy or light chain.

16 . The method of claim 1 , wherein the array is a bead array.

17 . The method of claim 1 , wherein the array is disposed on a slide.

18 . The method of claim 1 , wherein the biological sample is a tissue section.

19 . The method of claim 18 , wherein the tissue section is a fixed tissue section or a fresh frozen tissue section.

20 . The method of claim 19 , wherein the fixed tissue section is a formalin-fixed and paraffin-embedded (FFPE) tissue section.

Assignments (3)

Continuity (4)

References Cited (400)