METHODS OF SEQUENCING CIRCULAR TEMPLATE POLYNUCLEOTIDES
Disclosed herein, inter alia, are complexes, kits, and efficient methods of sequencing two strands of a double stranded polynucleotide.
1 .- 86 . (canceled)
87 . A cell comprising:
(a) a first concatemer and a second concatemer, wherein (i) the first concatemer comprises multiple copies of a first circular polynucleotide and the second concatemer comprises multiple complementary copies of a second circular polynucleotide, and (ii) the first concatemer and second concatemer are each immobilized to a cellular component or matrix within the cell;
(b) a first sequencing primer extension product hybridized to the first concatemer and a second sequencing primer extension product hybridized to the second concatemer, wherein each sequencing primer extension product comprises a nucleotide comprising a reversible terminator; and
(c) a polymerase bound to each sequencing primer extension product.
88 . The cell of claim 87 , wherein the first circular polynucleotide comprises single-stranded DNA or single-stranded RNA.
89 . The cell of claim 87 , wherein the first concatemer and second concatemer comprise a common sequence.
90 . The cell of claim 87 , wherein the first concatemer and second concatemer each comprise at least three copies of the circular polynucleotide.
91 . The cell of claim 87 , wherein the first circular polynucleotide comprises at least one cleavable site.
92 . The cell of claim 87 , further comprising an oligonucleotide capable of tethering each concatemer to a localized space in the cell.
93 . The cell of claim 92 , wherein the oligonucleotide comprises a blocking moiety at the 3′ end.
94 . The cell of claim 87 , wherein the nucleotide comprising a reversible terminator further comprises a detectable label.
95 . The cell of claim 87 , wherein the cellular component is a protein.
96 . The cell of claim 87 , wherein the matrix is a synthetic polymer matrix.
97 . The cell of claim 87 , wherein matrix is a swellable polymer or hydrogel.
98 . The cell of claim 87 , wherein the cell is immobilized on a solid support.
99 . The cell of claim 98 , wherein the solid support is located within a flow cell or microfluidic device.
100 . The cell of claim 98 , wherein the solid support further comprises a polymeric coating or hydrogel.
101 . The cell of claim 87 , wherein the cell is an immune cell.
102 . The cell of claim 87 , wherein the cell is a lung cancer cell, colorectal cancer cell, skin cancer cell, colon cancer cell, pancreatic cancer cell, breast cancer cell, cervical cancer cell, lymphoma cell, or leukemia cell.
103 . A tissue comprising:
(a) a first concatemer and a second concatemer, wherein (i) the first concatemer comprises multiple copies of a first circular polynucleotide and the second concatemer comprises multiple complementary copies of a second circular polynucleotide, and (ii) the first concatemer and second concatemer are each immobilized to a cellular component or matrix within a cell of the tissue;
(b) a first sequencing primer extension product hybridized to the first concatemer and a second primer extension product hybridized to the second concatemer, wherein each sequencing primer extension product comprises a nucleotide comprising a reversible terminator; and
(c) a polymerase bound to each sequencing primer extension product.
104 . The tissue of claim 103 , wherein the tissue is a formalin-fixed paraffin-embedded (FFPE) sample.
105 . The tissue of claim 103 , wherein the tissue is a skin tissue, muscle tissue, bone tissue, or organ tissue.