MULTIPLEXED TARGETED AMPLIFICATION OF POLYNUCLEOTIDES
Disclosed herein, inter alia, are compositions and methods for efficient amplification and sequencing of nucleic acid templates.
1 .- 51 . (canceled)
52 . A method of amplifying target polynucleotides, said method comprising:
contacting a solid support of with a first target polynucleotide comprising a first gene sequence and a second target polynucleotide comprising a second gene sequence;
hybridizing the first gene sequence to a first immobilized oligonucleotide of a first plurality of immobilized oligonucleotides, and extending with a polymerase the first immobilized oligonucleotide to generate a first extension product;
hybridizing the first extension product to a second immobilized oligonucleotide, and extending with a polymerase the second immobilized oligonucleotide to generate a second extension product; and
hybridizing the second extension product to a third immobilized oligonucleotide of the first plurality of immobilized oligonucleotides, and extending with a polymerase the third immobilized oligonucleotide to generate a third extension product; and
hybridizing the second gene sequence to a fourth immobilized oligonucleotide of a second plurality of immobilized oligonucleotides, and extending with a polymerase the fourth immobilized oligonucleotide to generate a fourth extension product;
hybridizing the fourth extension product to a fifth immobilized oligonucleotide, and extending with a polymerase the fifth immobilized oligonucleotide to generate a fifth extension product; and
hybridizing the fifth extension product to a sixth immobilized oligonucleotide of the second plurality of immobilized oligonucleotides, and extending with a polymerase the sixth immobilized oligonucleotide to generate a sixth extension product.
53 . The method of claim 52 , further comprising sequencing the first extension product, the second extension product, and/or the third extension product.
54 . The method of claim 52 , further comprising sequencing the second extension product and the third extension product.
55 . The method of claim 54 , further comprising sequencing the fifth extension product and the sixth extension product.
56 . The method of claim 52 , wherein the first gene sequence comprises a first exon sequence and the second gene sequence comprises a second exon sequence.
57 . The method of claim 52 , prior to contacting the solid support the target polynucleotides do not include a common primer binding sequence.
58 . The method of claim 52 , wherein the solid support is a particle.
59 . The method of claim 52 , wherein the first, second, and third, immobilized oligonucleotides are located in an area comprising a diameter of about 0.2 μm to about 2 μm.
60 . The method of claim 52 , wherein the first, second, third, fourth, fifth, and sixth immobilized oligonucleotides are located in an area comprising a diameter of about 0.2 μm to about 2 μm.
61 . The method of claim 52 , further comprising hybridizing a first sequencing primer to the second extension product and hybridizing a second sequencing primer to the fourth extension product, wherein the first sequencing primer and the second sequencing primer comprise the same sequence.
62 . The method of claim 52 , further comprising detecting the first gene sequence, or a complement thereof and detecting the second gene sequence, or complement thereof, wherein detecting comprises hybridizing a labeled oligonucleotide to each gene sequence and detecting the label.
63 . The method of claim 52 , wherein the first and second gene sequence each comprise a cancer-associated gene.
64 . The method of claim 63 , wherein the cancer associated gene is ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, or a VHL gene sequence.
65 . The method of claim 52 , further comprising performing bridge PCR to generate clusters of amplification products comprising the first gene sequence and the second gene sequence.
66 . The method of claim 52 , wherein contacting a solid support is performed under non-hybridizing conditions.
67 . The method of claim 52 , wherein the second immobilized oligonucleotide comprises, from 5′ to 3′, an amplification primer binding sequence, a barcode sequence, and a sequence complementary to the first extension product.
68 . A method of amplifying a target polynucleotide, said method comprising:
contacting a solid support with a target polynucleotide comprising a first gene sequence and a second gene sequence;
hybridizing the first gene sequence to a first immobilized oligonucleotide and extending with a polymerase the first immobilized oligonucleotide to generate a first extension product comprising a complement of the second gene sequence;
hybridizing the complement of the second gene sequence to a second immobilized oligonucleotide, and extending with a polymerase the second immobilized oligonucleotide to generate a second extension product; and
hybridizing the second extension product to a third immobilized oligonucleotide, and extending with a polymerase the third immobilized oligonucleotide to generate a third extension product.
69 . The method of claim 68 , wherein the second immobilized oligonucleotide comprises an amplification primer binding sequence and the second gene sequence.
70 . The method of claim 68 , further comprising sequencing the second and the third extension product.