Multiplexed sequencing in cells and tissues
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.
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 binding sequence of the first subset and sequencing a first sequence of the amplification products; followed by
hybridizing a sequencing primer complementary to the second sequencing primer binding sequence of the second subset and sequencing a second sequence of the amplification products; followed by
hybridizing a sequencing primer complementary to the third sequencing primer binding sequence of the third subset and sequencing a third sequence of the amplification products; followed by
hybridizing a sequencing primer complementary to the fourth sequencing primer binding sequence of the fourth subset and sequencing a fourth sequence of the amplification products; and
removing each sequencing primer prior to hybridizing a subsequent sequencing primer.