Multiplexed sequencing in cells and tissues

Patent metadata

Field	Value
Publication number	US-12590302-B2
Application number	US-202418672510-A
Country	US
Kind code	B2
Filing date	May 23, 2024
Priority date	Apr 27, 2021
Publication date	Mar 31, 2026
Grant date	Mar 31, 2026

Abstract

Disclosed herein, inter alia, are methods and compositions for detecting a plurality of nucleic acids in a sample including a cell or tissue. The methods may include amplifying nucleic acid molecules in the sample and detecting the amplified nucleic acid molecules in the sample.

First claim

What is claimed is:

1 . A method of detecting different nucleic acid molecules in a sample comprising a cell or tissue, said method comprising:
amplifying a plurality of different nucleic acid molecules in said cell or tissue to generate a first, a second, a third, and a fourth amplification product in said cell or tissue; binding a first primer to a first amplification product in said cell or tissue and incorporating 5 to 200 labeled nucleotides into the first primer and detecting the labeled nucleotides incorporated into the first primer; followed by binding a second primer to a second amplification product in said cell or tissue and incorporating 5 to 200 labeled nucleotides into the second primer and detecting the labeled nucleotides incorporated into the second primer; followed by binding a third primer to a third amplification product in said cell or tissue and incorporating 5 to 200 labeled nucleotides into the third primer and detecting the labeled nucleotides incorporated into the third primer; followed by binding a fourth primer to a fourth amplification product in said cell or tissue and incorporating 5 to 200 labeled nucleotides into the fourth primer and detecting the labeled nucleotides incorporated into the fourth primer; wherein the first, second, third, and fourth primers each comprise a different sequence.

2 . The method of claim 1 , wherein the different nucleic acid molecules are in a cell of said sample.

3 . The method of claim 1 , wherein the sample comprises liver tissue, kidney tissue, bone tissue, lung tissue, thymus tissue, adrenal tissue, skin tissue, bladder tissue, colon tissue, spleen tissue, or brain tissue.

4 . The method of claim 1 , wherein each amplification product comprises a gene sequence, or a complement thereof.

5 . The method of claim 1 , wherein amplifying comprises rolling circle amplification (RCA) or exponential rolling circle amplification (eRCA).

6 . The method of claim 1 , prior to binding the second primer, the method comprises contacting the first primer with a sequencing solution comprising one or more modified nucleotides comprising a reversible terminator, and monitoring the sequential incorporation of complementary nucleotides to generate a sequencing read, wherein the reversible terminator is removed prior to the introduction of the next complementary nucleotide.

7 . The method of claim 1 , wherein the method further comprises incorporating an irreversibly terminated nucleotide into the first primer prior to hybridizing the second primer.

8 . The method of claim 1 , wherein the first, second, third, and fourth primer are each a different sequence selected from SEQ ID NO:1 to SEQ ID NO:96.

9 . The method of claim 1 , wherein the plurality of different nucleic acid molecules comprises a first circular polynucleotide, a second circular polynucleotide, a third circular polynucleotide, and a fourth circular polynucleotide.

10 . The method of claim 9 , wherein each circular polynucleotide is synthetic and bound to an RNA molecule or DNA molecule in said sample.

11 . The method of claim 9 , wherein each circular polynucleotide is bound to an oligonucleotide attached to a protein-specific binding moiety.

12 . The method of claim 1 , wherein the plurality of nucleic acid molecules each comprise a barcode.

13 . The method of claim 1 , prior to amplifying, the method comprises:
binding a first polynucleotide probe comprising a 3′ and 5′ end to a first target polynucleotide and ligating the 5′ and 3′ ends together to form a first nucleic acid molecule; and binding a second polynucleotide probe comprising a 3′ and 5′ end to a second target polynucleotide and ligating the 5′ and 3′ ends together to form a second nucleic acid molecule; thereby forming different nucleic acid molecules.

14 . The method of claim 1 , wherein the first primer, second primer, third primer, and fourth primer are each bound to the amplification product simultaneously.

15 . The method of claim 1 , wherein the first primer, second primer, third primer, and fourth primer are each bound to the amplification product sequentially.

16 . The method of claim 1 , wherein the labeled nucleotides each comprise a fluorescent label and a reversible terminator.

17 . A method of detecting different nucleic acid molecules in a tissue sample, said method comprising:
amplifying a plurality of different circular polynucleotides in said tissue sample to generate a plurality of amplification products; binding a first primer to a first amplification product and incorporating a first labeled nucleotide into the first primer and incorporating an irreversibly terminated nucleotide into the first primer; binding a second primer to a second amplification product and incorporating a second labeled nucleotide into the second primer and incorporating an irreversibly terminated nucleotide into the second primer; and detecting the first and second incorporated nucleotides.

18 . The method of claim 17 , prior to detecting, the method comprises:
binding a third primer to a third amplification product and incorporating a third labeled nucleotide into the third primer and incorporating an irreversibly terminated nucleotide into the third primer; and binding a fourth primer to a fourth amplification product and incorporating a fourth labeled nucleotide into the fourth primer and incorporating an irreversibly terminated nucleotide into the fourth primer.

19 . The method of claim 18 , further comprising detecting the third and fourth labeled nucleotides.

20 . The method of claim 1 , wherein the sample comprises cancer cells.

21 . The method of claim 16 , wherein the reversible terminator comprises a C 2 -C 6 allyl moiety, a disulfide moiety, or an azido moiety.

22 . A method of detecting different nucleic acid molecules in a sample comprising a cell or tissue, said method comprising:
amplifying a plurality of different nucleic acid molecules in said sample to generate a plurality of amplification products in said sample; binding a first primer to a first amplification product and incorporating a first labeled nucleotide into the first primer and detecting the labeled nucleotide; binding a second primer to a second amplification product and incorporating a second labeled nucleotide into the second primer and detecting the second labeled nucleotide; binding a third primer to a third amplification product and incorporating a third labeled nucleotide into the third primer and detecting the third labeled nucleotide; and binding a fourth primer to a fourth amplification product and incorporating a fourth labeled nucleotide into the fourth primer and detecting the fourth labeled nucleotide; wherein the first, second, third, and fourth primer are each a different sequence selected from SEQ ID NO:1 to SEQ ID NO:96.

23 . A method of selectively sequencing nucleic acids in a cell or tissue, the method comprising:
generating a plurality of amplification products in the cell or tissue, wherein a first subset of the amplification products comprises a first primer binding sequence, a second subset of the amplification products comprises a second primer binding sequence, a third subset of the amplification products comprises a third primer binding sequence, and a fourth subset of the amplification products comprises a fourth primer binding sequence; hybridizing a sequencing primer complementary to the first sequencing primer bindin

Assignees

• Singular Genomics Systems Inc

Inventors

• Glezer Eli N
• Van Wietmarschen Niek
• Witters Daan

Classifications

• C12Q1/6837

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12N15/1068 Primary

  Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis · CPC title