US2022267841A1PendingUtilityA1
Adapters, methods, and compositions for duplex sequencing
Est. expiryDec 8, 2035(~9.4 yrs left)· nominal 20-yr term from priority
C12Q 1/6869C12Q 1/6855
73
PatentIndex Score
0
Cited by
0
References
0
Claims
Abstract
Disclosed herein are adapter nucleic acid sequences, double-stranded complexed nucleic acids, compositions, and methods for sequencing a double-stranded target nucleic acid with applications to error correction by duplex sequencing.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . A pair of adapter nucleic acid sequences for use in sequencing a double-stranded target nucleic acid molecule, comprising a first adapter nucleic acid sequence and a second adapter nucleic acid sequence, wherein each adapter nucleic acid sequence comprises:
a primer binding domain, a strand defining element (SDE), a single molecule identifier (SMI) domain, and a ligation domain; wherein the SDE of the first adapter nucleic acid sequence is at least partially non-complementary to the SDE of the second adapter nucleic acid sequence.
2 . The pair of adapter nucleic acid sequences of claim 1 , wherein the two adapter sequences consist of two separate DNA molecules that are at least partially annealed together.
3 . The pair of adapter nucleic acid sequences of claim 1 , wherein the first adapter nucleic acid sequence and the second adapter nucleic acid sequence are linked via a linker domain.
4 . The pair of adapter nucleic acid sequences of claim 3 , wherein the linker domain is comprised of nucleotides.
5 . The pair of adapter nucleic acid sequences of claim 3 , wherein the linker domain contains one or more modified nucleotide or non-nucleotide molecules.
6 . The pair of adapter nucleic acid sequences of claim 4 , wherein the one or more modified nucleotide or non-nucleotide molecule is selected from an abasic site; a uracil; tetrahydrofuran; 8-oxo-7,8-dihydro-2′-deoxyadenosine (8-oxo-A); 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-G); deoxyinosine, 5′-nitroindole; 5-Hydroxymethyl-2′-deoxycytidine; iso-cytosine; 5′-methyl-isocytosine; or iso-guanosine.
7 . The pair of adapter nucleic acid sequences of any one of claims 3 to 5 , wherein the linker domain forms a loop.
8 . The pair of adapter nucleic acid sequences of any one of claims 1 to 7 , wherein the SDE of the first adapter nucleic acid sequence is non-complementary to the SDE of the second adapter nucleic acid sequence.
9 . The pair of adapter nucleic acid sequences of any one of claims 1 to 7 , wherein the primer binding domain of the first adapter nucleic acid sequence is at least partially complementary to the primer binding domain of the second adapter nucleic acid sequence.
10 . The pair of adapter nucleic acid sequences of any one of claims 1 to 9 , wherein the primer binding domain of the first adapter nucleic acid sequence is complementary to the primer binding domain of the second adapter nucleic acid sequence.
11 . The pair of adapter nucleic acid sequences of any one of claims 1 to 7 , wherein the primer binding domain of the first adapter nucleic acid sequence is at least partially non-complementary to the primer binding domain of the second adapter nucleic acid sequence.
12 . The pair of adapter nucleic acid sequences of any one of claims 1 to 7 , wherein at least one SMI domain is an endogenous SMI.
13 . The pair of adapter nucleic acid sequences of claim 12 , wherein the endogenous SMI is related to a shear point.
14 . The pair of adapter nucleic acid sequences of any one of claims 1 to 11 , wherein the SMI domain comprises at least one degenerate or semi-degenerate nucleic acid.
15 . The pair of adapter nucleic acid sequences of any one of claims 1 to 11 , wherein the SMI domain is non-degenerate.
16 . The pair of adapter nucleic acid sequences of any one of claims 1 to 15 , wherein the sequence of the SMI domain is considered in conjunction with the sequence corresponding to randomly or semi-randomly sheared ends of ligated DNA to obtain an SMI sequence capable of distinguishing single DNA molecules from one another.
17 . The pair of adapter nucleic acid sequences of any one of claims 1 to 16 , wherein the SMI domain of the first adapter nucleic acid sequence is at least partially complementary to the SMI domain of the second adapter nucleic acid sequence.
18 . The pair of adapter nucleic acid sequences of any one of claims 1 to 17 , wherein the SMI domain of the first adapter nucleic acid sequence is complementary to the SMI domain of the second adapter nucleic acid sequence.
19 . The pair of adapter nucleic acid sequences of any one of claims 1 to 15 , wherein the SMI domain of the first adapter nucleic acid sequence is at least partially non-complementary to the SMI domain of the second adapter nucleic acid sequence.
20 . The pair of adapter nucleic acid sequences of claim 19 , wherein each SMI domain comprises a primer binding site.
21 . The pair of adapter nucleic acid sequences of claim 20 , wherein each SMI domain is located distal to its ligation domain.
22 . The pair of adapter nucleic acid sequences of claim 19 , wherein the SMI domain of the first adapter nucleic acid sequence is non-complementary to the SMI domain of the second adapter nucleic acid sequence.
23 . The pair of adapter nucleic acid sequences of any one of claims 1 to 22 , wherein each SMI domain comprises between about 1 to about 30 degenerate or semi-degenerate nucleic acids.
24 . The pair of adapter nucleic acid sequences of any one of claims 1 to 23 , wherein the ligation domain of the first adapter nucleic acid sequence is at least partially complementary to the ligation domain of the second adapter nucleic acid sequence.
25 . The pair of adapter nucleic acid sequences of any one of claims 1 to 24 , wherein each ligation domain is capable of being ligated to one strand of a double-stranded target nucleic acid sequence.
26 . The pair of adapter nucleic acid sequences of any one of claims 1 to 25 , wherein one of the ligation domains comprises a T-overhang, an A-overhang, a CG-overhang, a blunt end, or another ligateable nucleic acid sequence.
27 . The pair of adapter nucleic acid sequences of any one of claims 1 to 26 , wherein both ligation domains comprise a blunt end.
28 . The pair of adapter nucleic acid sequences of any claims 1 to 26 wherein at least one of the ligation domains comprises a modified nucleic acid.
29 . The pair of adapter nucleic acid sequences of claim 28 , wherein the modified nucleotide is selected from an abasic site; a uracil; tetrahydrofuran; 8-oxo-7,8-dihydro-2′-deoxyadenosine (8-oxo-A); 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-G); deoxyinosine, 5′-nitroindole; 5-Hydroxymethyl-2′-deoxycytidine; iso-cytosine; 5′-methyl-isocytosine; or iso-guanosine.
30 . The pair of adapter nucleic acid sequences of any claims 1 to 26 wherein at least one of the ligation domains comprises a dephosphorylated base.
31 . The pair of adapter nucleic acid sequences of any claims 1 to 26 wherein at least one of the ligation domains comprises a dehydroxylated base.
32 . The pair of adapter nucleic acid sequences of any claims 1 to 26 wherein at least one of the ligation domains has been chemically modified so as to render it unligateable.
33 . The pair of adapter nucleic acid sequences of any one of claims 1 to 27 , wherein the SDE of the first adapter nucleic acid sequence differs by and/or is non-complementary at at least one nucleotide from the SDE of the second adapter nucleic acid sequence.
34 . The pair of adapter nucleic acid sequences of any one of claims 1 to 27 , wherein at least one nucleotide is omitted from either the SDE of the first adapter nucleic acid sequence or from the SDE of the second adapter nucleic acid by an enzymatic reaction.
35 . The pair of adapter nucleic acid sequences of claim 34 , wherein the enzymatic reaction comprises a polymerase, an endonuclease, a glycosylase, or a lyase.
36 . The pair of adapter nucleic acid sequences of claim 33 , wherein the at least one nucleotide is a modified nucleotide or a nucleotide comprising a label.
37 . The pair of adapter nucleic acid sequences of claim 37 , wherein the modified nucleotide or a nucleotide comprising a label is selected from an abasic site; a uracil; tetrahydrofuran; 8-oxo-7,8-dihydro-2′-deoxyadenosine (8-oxo-A); 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-G); deoxyinosine, 5′-nitroindole; 5-Hydroxymethyl-2′-deoxycytidine; iso-cytosine; 5′-methyl-isocytosine; or iso-guanosine.
38 . The pair of adapter nucleic acid sequences of claim 35 , wherein the SDE of the first adapter nucleic acid sequence comprises a self-complementary domain that is capable of forming a hairpin loop.
39 . The pair of adapter nucleic acid sequences of any one of claims 1 to 38 , wherein the end of first adapter nucleic acid sequence distal to its ligation domain is ligated to the end of the second adapter nucleic acid sequence that is distal to its ligation domain, thereby forming a loop.
40 . The pair of adapter nucleic acid sequences of claim 39 , wherein the loop comprises a restriction enzyme recognition site.
41 . The pair of adapter nucleic acid sequences of any one of claims 1 to 35 , wherein at least the first adapter nucleic acid sequence further comprises a second SDE.
42 . The pair of adapter nucleic acid sequences of claim 41 , wherein the second SDE is located at a terminus of the first adapter nucleic acid sequence.
43 . The pair of adapter nucleic acid sequences of claim 41 or claim 42 , wherein the second adapter nucleic acid sequence further comprises a second SDE.
44 . The pair of adapter nucleic acid sequences of any one of claims 41 to 43 , wherein the second SDE is located at a terminus of the second adapter nucleic acid sequence.
45 . The pair of adapter nucleic acid sequences of any one of claims 41 to 44 , wherein the second SDE of the first adapter nucleic acid sequence is at least partially non-complementary to the second SDE of the second adapter nucleic acid sequence.
46 . The pair of adapter nucleic acid sequences of claim 45 , wherein the second SDE of the first adapter nucleic acid sequence differs by and/or is non-complementary at at least one nucleotide from the second SDE of the second adapter nucleic acid sequence.
47 . The pair of adapter nucleic acid sequences of claim 46 , wherein at least one nucleotide is omitted from either the second SDE of the first adapter nucleic acid sequence or from the second SDE of the second adapter nucleic acid by an enzymatic reaction.
48 . The pair of adapter nucleic acid sequences of claim 47 , wherein the enzymatic reaction comprises a polymerase, an endonuclease, a glycosylase, or a lyase.
49 . The pair of adapter nucleic acid sequences of claim 45 or claim 46 , wherein the second SDE of the first adapter nucleic acid sequence is non-complementary to the second SDE of the second adapter nucleic acid sequence.
50 . The pair of adapter nucleic acid sequences of any one of claims 41 to 45 , wherein the SDE of the first adapter nucleic acid sequence is directly linked to the second SDE of the second adapter nucleic acid sequence.
51 . The pair of adapter nucleic acid sequences of any one of claims 1 to 50 , wherein the primer binding domain of the first adapter nucleic acid sequence is located 5′ to a first SDE.
52 . The pair of adapter nucleic acid sequences of any one of claims 1 to 51 , wherein the first SDE of the first adapter nucleic acid sequence is located 5′ to the SMI domain.
53 . The pair of adapter nucleic acid sequences of any one of claims 1 to 51 , wherein the first SDE of the first adapter nucleic acid sequence is located 3′ to the SMI domain.
54 . The pair of adapter nucleic acid sequences of any one of claims 1 to 53 , wherein the first SDE of the first adapter nucleic acid sequence is located 5′ to the SMI domain and is located 3′ to the primer binding domain.
55 . The pair of adapter nucleic acid sequences of any one of claims 1 to 53 , wherein the first SDE of the first adapter nucleic acid sequence is located 3′ to the SMI domain which is located 3′ to the primer binding domain.
56 . The pair of adapter nucleic acid sequences of any one of claims 1 to 54 , wherein the SMI domain of the first adapter nucleic acid sequence is located 5′ to the ligation domain.
57 . The pair of adapter nucleic acid sequences of any one of claims 1 to 56 , wherein the 3′ terminus of the first adapter nucleic acid sequence comprises the ligation domain.
58 . The pair of adapter nucleic acid sequences of any one of claims 1 to 57 , wherein the first adapter nucleic acid sequence comprises, from 5′ to 3′, the primer binding domain, the first SDE, the SMI domain, and the ligation domain.
59 . The pair of adapter nucleic acid sequences of any one of claims 1 to 57 , wherein the first adapter nucleic acid sequence comprises, from 5′ to 3′, the primer binding domain, the SMI domain, the first SDE, and the ligation domain.
60 . The pair of adapter nucleic acid sequences of any one of claims 1 to 59 , wherein either the first adapter nucleic acid sequence or the second adapter nucleic acid sequence comprises a modified nucleotide or a non-nucleotide molecule.
61 . The pair of adapter nucleic acid sequences of claim 60 , wherein the modified nucleotide or non-nucleotide molecule is Colicin E2, Im2, Glutithione, glutathione-s-transferase (GST), Nickel, poly-histidine, FLAG-tag, myc-tag, or biotin.
62 . The pair of adapter nucleic acid sequences of claim 61 , wherein the biotin is Biotin-16-Aminoallyl-2′-deoxyuridine-5′-Triphosphate, Biotin-16-Aminoallyl-2′-deoxycytidine-5′-Triphosphate, Biotin-16-Aminoallylcytidine-5′-Triphosphate, N4-Biotin-OBEA-2′-deoxycytidine-5′-Triphosphate, Biotin-16-Aminoallyluridine-5′-Triphosphate, Biotin-16-7-Deaza-7-Aminoallyl-2′-deoxyguanosine-5′-Triphosphate, Desthiobiotin-6-Aminoallyl-2′-deoxycytidine-5′-Triphosphate, 5′-Biotin-G-Monophosphate, 5′-Biotin-A-Monophosphate, 5′-Biotin-dG-Monophosphate, or 5′-Biotin-dA-Monophosphate.
63 . The pair of adapter nucleic acid sequences of claim 61 or claim 62 , wherein the biotin is capable of being bound to a streptavidin attached to a substrate.
64 . The pair of adapter nucleic acid sequences of claim 63 , wherein when the biotin is bound to a streptavidin attached to a substrate, the first adapter nucleic acid sequence is capable of separating from the second adapter nucleic acid sequence.
65 . The pair of adapter nucleic acid sequences of any one of claims 1 to 59 , wherein either the first adapter nucleic acid sequence or the second adapter nucleic acid sequence comprises an affinity label selected from a small molecule, a nucleic acid, a peptide, and a uniquely bindeable moiety which is capable of being bound by an affinity partner.
66 . The pair of adapter nucleic acid sequences of claim 65 , wherein when the affinity partner is attached to a solid substrate and bound to the affinity label the adapter nucleic acid sequence comprising the affinity label is capable of being separated from the adapter nucleic acid sequence not comprising the affinity label.
67 . The pair of adapter nucleic acid sequences of claim 66 , wherein the solid substrate is a solid surface, a bead, or another fixed structure.
68 . The pair of adapter nucleic acid sequences of any one of claims 65 to 67 , wherein the nucleic acid is DNA, RNA, or a combination thereof, and optionally, comprising a peptide-nucleic acid or a locked nucleic acid.
69 . The pair of adapter nucleic acid sequences of any one of claims 65 to 68 , wherein the affinity label is located at a terminus of an adapter or within a domain in the first adapter nucleic acid sequence that is not completely complementary to an opposing domain in the second adapter nucleic acid sequence.
70 . The pair of adapter nucleic acid sequences of any one of claims 1 to 59 , wherein either the first adapter nucleic acid sequence or the second adapter nucleic acid sequence comprises a physical group having a magnetic property, a charge property, or an insolubility property.
71 . The pair of adapter nucleic acid sequences of claim 70 , wherein when the physical group has a magnetic property and a magnetic field is applied, the adapter nucleic acid sequence comprising the physical group is separated from the adapter nucleic acid sequence not comprising the physical group.
72 . The pair of adapter nucleic acid sequences of claim 70 , wherein when the physical group has a charge property and an electric field is applied, the adapter nucleic acid sequence comprising the physical group is separated from the adapter nucleic acid sequence not comprising the physical group.
73 . The pair of adapter nucleic acid sequences of claim 70 , wherein when the physical group has an insolubility property and the pair of adapter nucleic acid sequences are contained in a solution for which the physical group is insoluble, the adapter nucleic acid sequence comprising the physical group is precipitated away from the adapter nucleic acid sequence not comprising the physical group which remains in solution.
74 . The pair of adapter nucleic acid sequences of any one of claims 70 to 73 , wherein the physical group is located at a terminus of an adapter or within a domain in the first adapter nucleic acid sequence that is not completely complementary to an opposing domain in the second adapter nucleic acid sequence.
75 . The pair of adapter nucleic acid sequences of any one of claims 1 to 64 , wherein the second adapter nucleic acid sequence comprises at least one phosphorothioate bond.
76 . The pair of adapter nucleic acid sequences of any one of claims 1 to 75 , wherein the double-stranded target nucleic acid sequence is DNA or RNA.
77 . The pair of adapter nucleic acid sequences of any one of claims 1 to 76 , wherein each adapter nucleic acid sequences comprises a ligation domain at each of its termini.
78 . The pair of adapter nucleic acid sequences of any one of claims 1 to 77 , wherein the first adapter nucleic acid sequence or the second adapter nucleic acid sequence is at least partially single-stranded.
79 . The pair of adapter nucleic acid sequences of claim 78 , wherein the first adapter nucleic acid sequence or the second adapter nucleic acid sequence is single-stranded.
80 . The pair of adapter nucleic acid sequences of claim 79 , wherein the first adapter nucleic acid sequence and the second adapter nucleic acid sequence is single-stranded.
81 . A composition comprising at least one pair of adapter nucleic acid sequences of any of the proceeding claims and a second pair of adapter nucleic acid sequences, wherein each strand of the second pair of adapter nucleic acid sequences comprises at least a primer binding site and a ligation domain.
82 . The composition of claim 81 , further comprising an SMI domain in each strand of the second pair of adapter nucleic acid sequence.
83 . The composition of claim 81 or claim 82 further comprising a primer binding site in each strand of the second pair of adapter nucleic acid sequence.
84 . A composition comprising at least two pairs of adapter nucleic acid sequences of any of the proceeding claims, wherein the SDE of a first adapter nucleic acid sequence from a first pair of adapter nucleic acid sequences differs from the SDE of a first adapter nucleic acid sequence from at least a second pair of adapter nucleic acid sequences.
85 . A composition comprising at least two pairs of adapter nucleic acid molecules of any of any one of claims 1 to 80 , wherein the SMI domain of a first adapter nucleic acid molecule from a first pair of adapter nucleic acid molecules differs from the SMI domain of a first adapter nucleic acid molecule from an at least second pair of adapter nucleic acid molecules.
86 . The composition of claim 85 , wherein the SMI domain of the first adapter nucleic acid molecule from the first pair of single-stranded adapter nucleic acid molecules is the same length as the SMI domain of the first single-stranded adapter nucleic acid molecule from the at least second pair of single-stranded adapter nucleic acid molecules.
87 . The composition of claim 85 , wherein the SMI domain of the first adapter nucleic acid molecule from the first pair of single-stranded adapter nucleic acid molecules has a different length than the SMI domain of the first single-stranded adapter nucleic acid molecule from the at least second pair of single-stranded adapter nucleic acid molecules.
88 . The composition of any one of claims 85 to 87 , wherein each SMI domain comprises one or more fixed bases at a site within or flanking the SMI.
89 . A composition comprising at least a first double-stranded complexed nucleic acid comprising a first pair of adapter nucleic acid molecules of any one of claims 1 to 88 ligated to a first terminus of a double-stranded target nucleic acid molecule and a second pair of adapter nucleic acid molecules of any one of claims 1 to 50 ligated to a second terminus of the double-stranded target nucleic acid molecule.
90 . The composition of claim 89 , wherein the first pair of adapter nucleic acid molecules is different from the second pair of adapter nucleic acid molecules.
91 . The composition of claim 90 , wherein the first strand adapter-target nucleic acid molecule of the first pair of adapter nucleic acid molecules comprises a first SMI domain and the first strand adapter-target nucleic acid molecule of the second pair of adapter nucleic acid molecules comprises a second SMI domain.
92 . The composition of any one of claims 89 to 91 comprising at least a second double-stranded complexed nucleic acid.
93 . A pair of adapter nucleic acid sequences for use in sequencing a double-stranded target nucleic acid molecule, comprising a first adapter nucleic acid sequence and a second adapter nucleic acid sequence, wherein each adapter nucleic acid sequence comprises:
a primer binding domain, and a single molecule identifier (SMI) domain.
94 . The pair of adapter nucleic acid sequences of claim 93 , wherein at least one of the first adapter nucleic acid sequence or the second adapter nucleic acid sequence further comprises a domain comprising at least one modified nucleotide.
95 . The pair of adapter nucleic acid sequences of claim 93 or claim 94 , wherein the first adapter nucleic acid sequence and the second adapter nucleic acid sequence further comprise a domain comprising at least one modified nucleotide.
96 . The pair of adapter nucleic acid sequences of any one of claims 93 to 95 , wherein at least one of the first adapter nucleic acid sequence or the second adapter nucleic acid sequence further comprises a ligation domain.
97 . The pair of adapter nucleic acid sequences of any one of claims 93 to 95 , wherein the first adapter nucleic acid sequence and the second adapter nucleic acid sequence comprises a ligation domain.
98 . The pair of adapter nucleic acid sequences of claim 94 to claim 97 , wherein the at least one modified nucleotide is selected from an abasic site; a uracil; tetrahydrofuran; 8-oxo-7,8-dihydro-2′-deoxyadenosine (8-oxo-A); 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-G); deoxyinosine, 5′-nitroindole; 5-Hydroxymethyl-2′-deoxycytidine; iso-cytosine; 5′-methyl-isocytosine; or iso-guanosine.
99 . The pair of adapter nucleic acid sequences of any one of claim 97 to claim 98 , wherein the two adapter sequences consist of two separate DNA molecules that are at least partially annealed together.
100 . The pair of adapter nucleic acid sequences of claim 97 , wherein the first adapter nucleic acid sequence and the second adapter nucleic acid sequence are linked via a linker domain.
101 . The pair of adapter nucleic acid sequences of claim 100 , wherein the linker domain is comprised of nucleotides.
102 . The pair of adapter nucleic acid sequences of claim 100 , wherein the linker domain contains one or more modified nucleotide or non-nucleotide molecules.
103 . The pair of adapter nucleic acid sequences of claim 102 , wherein at least one modified nucleotide or non-nucleotide molecule is selected from an abasic site; a uracil; tetrahydrofuran; 8-oxo-7,8-dihydro-2′-deoxyadenosine (8-oxo-A); 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-G); deoxyinosine, 5′-nitroindole; 5-Hydroxymethyl-2′-deoxycytidine; iso-cytosine; 5′-methyl-isocytosine; or iso-guanosine.
104 . The pair of adapter nucleic acid sequences of any one of claims 58 to 61 , wherein the linker domain forms a loop.
105 . The pair of adapter nucleic acid sequences of any one of claims 97 to 104 , wherein the primer binding domain of the first adapter nucleic acid sequence is at least partially complementary to the primer binding domain of the second adapter nucleic acid sequence.
106 . The pair of adapter nucleic acid sequences of any one of claims 97 to 104 , wherein the primer binding domain of the first adapter nucleic acid sequence is complementary to the primer binding domain of the second adapter nucleic acid sequence.
107 . The pair of adapter nucleic acid sequences of any one of claims 97 to 104 , wherein the primer binding domain of the first adapter nucleic acid sequence is non-complementary to the primer binding domain of the second adapter nucleic acid sequence.
108 . The pair of adapter nucleic acid sequences of any one of claims 97 to 107 , wherein at least one SMI domain is an endogenous SMI.
109 . The pair of adapter nucleic acid sequences of claim 108 , wherein the endogenous SMI is related to a shear point.
110 . The pair of adapter nucleic acid sequences of any one of claims 97 to 109 , wherein the SMI domain comprises at least one degenerate or semi-degenerate nucleic acid.
111 . The pair of adapter nucleic acid sequences of any one of claims 97 to 110 , wherein the SMI domain is non-degenerate.
112 . The pair of adapter nucleic acid sequences of any one of claims 97 to 111 , wherein the sequence of the SMI domain is considered in conjunction with the sequence corresponding to randomly or semi-randomly sheared ends of ligated DNA to obtain an SMI sequence capable of distinguishing single DNA molecules from one another.
113 . The pair of adapter nucleic acid sequences of any one of claims 97 to 112 , wherein the SMI domain of the first adapter nucleic acid sequence is at least partially complementary to the SMI domain of the second adapter nucleic acid sequence.
114 . The pair of adapter nucleic acid sequences of any one of claims 97 to 113 , wherein the SMI domain of the first adapter nucleic acid sequence is complementary to the SMI domain of the second adapter nucleic acid sequence.
115 . The pair of adapter nucleic acid sequences of any one of claims 97 to 113 , wherein the SMI domain of the first adapter nucleic acid sequence is at least partially non-complementary to the SMI domain of the second adapter nucleic acid sequence.
116 . The pair of adapter nucleic acid sequences of any one of claims 97 to 113 , wherein the SMI domain of the first adapter nucleic acid sequence is non-complementary to the SMI domain of the second adapter nucleic acid sequence.
117 . The pair of adapter nucleic acid sequences of any one of claims 97 to 116 , wherein each SMI domain comprises between about 1 to about 30 degenerate or semi-degenerate nucleic acids.
118 . The pair of adapter nucleic acid sequences of any one of claims 97 to 117 , wherein the ligation domain of the first adapter nucleic acid sequence is at least partially complementary to the ligation domain of the second adapter nucleic acid sequence.
119 . The pair of adapter nucleic acid sequences of any one of claims 97 to 118 , wherein each ligation domain is capable of being ligated to one strand of a double-stranded target nucleic acid sequence.
120 . The pair of adapter nucleic acid sequences of any one of claims 97 to 119 , wherein one of the ligation domains comprises a T-overhang, an A-overhang, a CG-overhang, a blunt end, or another ligateable nucleic acid sequence.
121 . The pair of adapter nucleic acid sequences of any one of claims 97 to 120 , wherein both ligation domains comprise a blunt end.
122 . The pair of adapter nucleic acid sequences of any one of claims 97 to 121 , wherein each SMI domain comprises a primer binding site.
123 . The pair of adapter nucleic acid sequences of any one of claims 97 to 122 , wherein at least the first adapter nucleic acid sequence further comprises an SDE.
124 . The pair of adapter nucleic acid sequences of claim 123 , wherein the SDE is located at a terminus of the first adapter nucleic acid sequence.
125 . The pair of adapter nucleic acid sequences of claim 123 or claim 124 , wherein the second adapter nucleic acid sequence further comprises an SDE.
126 . The pair of adapter nucleic acid sequences of any one of claims 123 to 124 , wherein the SDE is located at a terminus of the second adapter nucleic acid sequence.
127 . The pair of adapter nucleic acid sequences of any one of claims 123 to 126 , wherein the SDE of the first adapter nucleic acid sequence is at least partially non-complementary to the SDE of the second adapter nucleic acid sequence.
128 . The pair of adapter nucleic acid sequences of claim 127 , wherein the SDE of the first adapter nucleic acid sequence is non-complementary to the SDE of the second adapter nucleic acid sequence.
129 . The pair of adapter nucleic acid sequences of any one of claims 123 to 128 , wherein the SDE of the first adapter nucleic acid sequence is directly linked to the SDE of the second adapter nucleic acid sequence.
130 . The pair of adapter nucleic acid sequences of any one of claims 123 to 129 , wherein the SDE of the first adapter nucleic acid sequence differs by and/or is non-complementary at at least one nucleotide from the SDE of the second adapter nucleic acid sequence.
131 . The pair of adapter nucleic acid sequences of claim 130 , wherein the least one nucleotide is omitted from either the SDE of the first adapter nucleic acid sequence or from the SDE of the second adapter nucleic acid by an enzymatic reaction.
132 . The pair of adapter nucleic acid sequences of claim 131 , wherein the enzymatic reaction comprises a polymerase or an endonuclease.
133 . The pair of adapter nucleic acid sequences of claim 132 , wherein the at least one nucleotide is a modified nucleotide or a nucleotide comprising a label.
134 . The pair of adapter nucleic acid sequences of claim 134 , wherein the modified nucleotide or a nucleotide comprising a label is selected from an abasic site; a uracil; tetrahydrofuran; 8-oxo-7,8-dihydro-2′-deoxyadenosine (8-oxo-A); 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-G); deoxyinosine, 5′-nitroindole; 5-Hydroxymethyl-2′-deoxycytidine; iso-cytosine; 5′-methyl-isocytosine; or iso-guanosine.
135 . The pair of adapter nucleic acid sequences of any one of claims 123 to 135 , wherein the SDE of the first adapter nucleic acid sequence comprises a self-complementary domain that is capable of forming a hairpin loop.
136 . The pair of adapter nucleic acid sequences of any one of claims 123 to 135 , wherein the end of first adapter nucleic acid sequence distal to its ligation domain is ligated to the end of the second adapter nucleic acid sequence that is distal to its ligation domain, thereby forming a loop.
137 . The pair of adapter nucleic acid sequences of claim 136 , wherein the loop comprises a restriction enzyme recognition site.
138 . The pair of adapter nucleic acid sequences of any one of claims 93 to 129 , wherein the primer binding domain of the first adapter nucleic acid sequence is located 5′ to the SMI domain.
139 . The pair of adapter nucleic acid sequences of any one of claims 93 to 138 , wherein the domain comprising at least one modified nucleotide of the first adapter nucleic acid sequence is located 5′ to the SMI domain.
140 . The pair of adapter nucleic acid sequences of any one of claims 93 to 138 wherein the domain comprising at least one modified nucleotide of the first adapter nucleic acid sequence is located 3′ to the SMI domain.
141 . The pair of adapter nucleic acid sequences of any one of claims 93 to 139 , wherein the domain comprising at least one modified nucleotide of the first adapter nucleic acid sequence is located 5′ to the SMI domain and is located 3′ to the primer binding domain.
142 . The pair of adapter nucleic acid sequences of any one of claims 93 to 138 , wherein the domain comprising at least one modified nucleotide of the first adapter nucleic acid sequence is located 3′ to the SMI domain which is located 3′ to the primer binding domain.
143 . The pair of adapter nucleic acid sequences of any one of claims 93 to 141 , wherein the SMI domain of the first adapter nucleic acid sequence is located 5′ to the ligation domain.
144 . The pair of adapter nucleic acid sequences of any one of claims 93 to 143 , wherein the 3′ terminus of the first adapter nucleic acid sequence comprises the ligation domain.
145 . The pair of adapter nucleic acid sequences of any one of claims 93 to 143 , wherein the first adapter nucleic acid sequence comprises, from 5′ to 3′, the primer binding domain, the domain comprising at least one modified nucleotide, the SMI domain, and the ligation domain.
146 . The pair of adapter nucleic acid sequences of any one of claims 93 to 138 , wherein the first adapter nucleic acid sequence comprises, from 5′ to 3′, the primer binding domain, the SMI domain, the domain comprising at least one modified nucleotide, and the ligation domain.
147 . The pair of adapter nucleic acid sequences of any one of claims 93 to 146 , wherein either the first adapter nucleic acid sequence or the second adapter nucleic acid sequence comprises a modified nucleotide or a non-nucleotide molecule.
148 . The pair of adapter nucleic acid sequences of claim 147 , wherein the modified nucleotide or non-nucleotide molecule is Colicin E2, Im2, Glutithione, glutathione-s-transferase (GST), Nickel, poly-histidine, FLAG-tag, myc-tag, or biotin.
149 . The pair of adapter nucleic acid sequences of claim 148 , wherein biotin is Biotin-16-Aminoallyl-2′-deoxyuridine-5′-Triphosphate, Biotin-16-Aminoallyl-2′-deoxycytidine-5′-Triphosphate, Biotin-16-Aminoallylcytidine-5′-Triphosphate, N4-Biotin-OBEA-2′-deoxycytidine-5′-Triphosphate, Biotin-16-Aminoallyluridine-5′-Triphosphate, Biotin-16-7-Deaza-7-Aminoallyl-2′-deoxyguanosine-5′-Triphosphate, Desthiobiotin-6-Aminoallyl-2′-deoxycytidine-5′-Triphosphate, 5′-Biotin-G-Monophosphate, 5′-Biotin-A-Monophosphate, 5′-Biotin-dG-Monophosphate, or 5′-Biotin-dA-Monophosphate.
150 . The pair of adapter nucleic acid sequences of claim 148 or claim 149 , wherein the biotin is capable of being bound to a streptavidin attached to a substrate.
151 . The pair of adapter nucleic acid sequences of claim 150 , wherein when the biotin is bound to a streptavidin attached to a substrate, the first adapter nucleic acid sequence is capable of separating from the second adapter nucleic acid sequence.
152 . The pair of adapter nucleic acid sequences of any one of claims 93 to 148 , wherein the second adapter nucleic acid sequence comprises at least one phosphorothioate bond.
153 . The pair of adapter nucleic acid sequences of any one of claims 93 to 152 , wherein the double-stranded target nucleic acid sequence is DNA or RNA.
154 . The pair of adapter nucleic acid sequences of any one of claims 93 to 153 , wherein either the first adapter nucleic acid sequence or the second adapter nucleic acid sequence comprises an affinity label selected from a small molecule, a nucleic acid, a peptide, and a uniquely bindeable moiety which is capable of being bound by an affinity partner.
155 . The pair of adapter nucleic acid sequences of claim 154 wherein when the affinity partner is attached to a solid substrate and bound to the affinity label the adapter nucleic acid sequence comprising the affinity label is capable of being separated from the adapter nucleic acid sequence not comprising the affinity label.
156 . The pair of adapter nucleic acid sequences of claim 155 , wherein the solid substrate is a solid surface, a bead, or another fixed structure.
157 . The pair of adapter nucleic acid sequences of any one of claims 154 to 156 , wherein the nucleic acid is DNA, RNA, or a combination thereof, and optionally, comprising a peptide-nucleic acid or a locked nucleic acid.
158 . The pair of adapter nucleic acid sequences of any one of claims 154 to 157 , wherein the affinity label is located at a terminus of an adapter or within a domain in the first adapter nucleic acid sequence that is not completely complementary to an opposing domain in the second adapter nucleic acid sequence.
159 . The pair of adapter nucleic acid sequences of any one of claims 93 to 158 , wherein either the first adapter nucleic acid sequence or the second adapter nucleic acid sequence comprises a physical group having a magnetic property, a charge property, or an insolubility property.
160 . The pair of adapter nucleic acid sequences of claim 159 , wherein when the physical group has a magnetic property and a magnetic field is applied, the adapter nucleic acid sequence comprising the physical group is separated from the adapter nucleic acid sequence not comprising the physical group.
161 . The pair of adapter nucleic acid sequences of claim 159 , wherein when the physical group has a charge property and an electric field is applied, the adapter nucleic acid sequence comprising the physical group is separated from the adapter nucleic acid sequence not comprising the physical group.
162 . The pair of adapter nucleic acid sequences of claim 157 , wherein when the physical group has an insolubility property and the pair of adapter nucleic acid sequences are contained in a solution for which the physical group is insoluble, the adapter nucleic acid sequence comprising the physical group is precipitated away from the adapter nucleic acid sequence not comprising the physical group which remains in solution.
163 . The pair of adapter nucleic acid sequences of any one of claims 157 to 162 , wherein the physical group is located at a terminus of an adapter or within a domain in the first adapter nucleic acid sequence that is not completely complementary to an opposing domain in the second adapter nucleic acid sequence.
164 . The pair of adapter nucleic acid sequences of any one of claims 93 to 163 , wherein the first adapter nucleic acid sequence or the second adapter nucleic acid sequence is at least partially single-stranded.
165 . The pair of adapter nucleic acid sequences of claim 163 wherein the first adapter nucleic acid sequence or the second adapter nucleic acid sequence is single-stranded.
166 . The pair of adapter nucleic acid sequences of claim 165 , wherein the first adapter nucleic acid sequence and the second adapter nucleic acid sequence is single-stranded.
167 . The pair of adapter nucleic acid sequences of any one of claims 96 to 166 , wherein at least one of the ligation domains comprises a dehydroxylated base.
168 . The pair of adapter nucleic acid sequences of any one of claims 96 to 166 , wherein at least one of the ligation domains has been chemically modified so as to render it unligateable.
169 . A composition comprising at least two pairs of adapter nucleic acid molecules of any of any one of claims 93 to 168 , wherein the SMI domain of a first adapter nucleic acid molecule from a first pair of adapter nucleic acid molecules differs from the SMI domain of a first adapter nucleic acid molecule from an at least second pair of adapter nucleic acid molecules.
170 . The composition of claim 169 , wherein the SMI domain of the first adapter nucleic acid molecule from the first pair of single-stranded adapter nucleic acid molecules is the same length as the SMI domain of the first single-stranded adapter nucleic acid molecule from the at least second pair of single-stranded adapter nucleic acid molecules.
171 . The composition of claim 169 , wherein the SMI domain of the first adapter nucleic acid molecule from the first pair of single-stranded adapter nucleic acid molecules has a different length than the SMI domain of the first single-stranded adapter nucleic acid molecule from the at least second pair of single-stranded adapter nucleic acid molecules.
172 . The composition of any one of claims 169 to 171 , wherein each SMI domain comprises one or more fixed bases at a site within or flanking the SMI.
173 . A composition comprising at least a first double-stranded complexed nucleic acid comprising a first pair of adapter nucleic acid molecules of any one of claims 93 to 168 ligated to a first terminus of a double-stranded target nucleic acid molecule and a second pair of adapter nucleic acid molecules of any one of claims 93 to 168 ligated to a second terminus of the double-stranded target nucleic acid molecule.
174 . The composition of claim 173 , wherein the first pair of adapter nucleic acid molecules is different from the second pair of adapter nucleic acid molecules.
175 . The composition of claim 174 , wherein the first strand adapter-target nucleic acid molecule of the first pair of adapter nucleic acid molecules comprises a first SMI domain and the first strand adapter-target nucleic acid molecule of the second pair of adapter nucleic acid molecules comprises a second SMI domain.
176 . The composition of claim 175 , wherein the first strand adapter-target nucleic acid molecule of the first pair of adapter nucleic acid molecules comprises a first SMI domain and the first strand adapter-target nucleic acid molecule of the second pair of adapter nucleic acid molecules comprises a second SMI domain.
177 . The composition of any one of claims 173 to 176 comprising at least a second double-stranded complexed nucleic acid.
178 . A composition comprising at least one pair of adapter nucleic acid molecules of any of any one of claims 1 to 80 and at least one pair of adapter nucleic acid molecules of any of any one of claims 93 to 168 .
179 . A composition comprising at least a first double-stranded complexed nucleic acid comprising a first pair of adapter nucleic acid molecules of any one of claims 1 to 80 ligated to a first terminus of a double-stranded target nucleic acid molecule and a second pair of adapter nucleic acid molecules of any one of claims 93 to 168 ligated to a second terminus of the double-stranded target nucleic acid molecule.
180 . A method of sequencing a double-stranded target nucleic acid comprising steps of:
(1) ligating a pair of adapter nucleic acid sequences of any one of claims 1 to 80 to at least one terminus of a double-stranded target nucleic acid molecule, thereby forming a double-stranded nucleic acid molecule comprising a first strand adapter-target nucleic acid sequence and a second strand adapter-target nucleic acid sequence; (2) amplifying the first strand adapter-target nucleic acid sequence, thereby producing a first set of amplified products comprising a plurality of first strand adapter-target nucleic acid sequences and a plurality of its complementary molecules; (3) amplifying the second strand adapter-target nucleic acid sequence, thereby producing a second set of amplified products comprising a plurality of second strand adapter-target nucleic acid sequences and a plurality of its complementary molecules, wherein the second set of amplified products is distinguishable from the first set of amplified products; (4) sequencing the first set of amplified products; and (5) sequencing the second set of amplified products.
181 . The method of claim 180 wherein the at least one terminus is two termini.
182 . The method of claim 180 or claim 181 , wherein amplification is performed by PCR.
183 . The method of claim 180 , wherein amplification is performed by multiple displacement amplification.
184 . The method of claim 180 , wherein amplification is performed by isothermal amplification.
185 . The method of claims 180 to 184 , wherein the pair of adapter nucleic acid sequences ligated to a first terminus of the double-stranded target nucleic acid sequence has an identical structure to the pair of adapter nucleic acid sequences ligated to a second terminus of the double-stranded target nucleic acid sequence.
186 . The method of claim 180 or claim 185 , wherein the first strand adapter-target nucleic acid sequence comprises in 5′ to 3′ order:
(a) a first adapter nucleic acid sequence;
(b) a first strand of the double-stranded target nucleic acid; and
(c) a second adapter nucleic acid sequence.
187 . The method of any one of claims 180 to 186 , wherein the second strand adapter-target nucleic acid sequence comprises in 3′ to 5′ order:
(a) a first adapter nucleic acid sequence;
(b) a second strand of the double-stranded target nucleic acid; and
(c) a second adapter nucleic acid sequence.
188 . The method of claim 180 , wherein the pair of adapter nucleic acid sequences ligated to a first terminus of the double-stranded target nucleic acid sequence is different from the pair of adapter nucleic acid sequences ligated to a second terminus of the double-stranded target nucleic acid sequence.
189 . The method of claim 188 , wherein the pair of adapter nucleic acid sequences ligated to a first terminus of the double-stranded target nucleic acid sequence has a first SMI domain and the pair of adapter nucleic acid sequences ligated to a second terminus of the double-stranded target nucleic acid sequence has a second SMI domain;
wherein the first SMI domain may be different from the second SMI domain.
190 . The method of claim 188 or claim 189 , wherein the first strand adapter-target nucleic acid sequence comprises in 5′ to 3′ order:
(a) a first adapter nucleic acid sequence comprising the first SDE;
(b) a first SMI domain;
(c) a first strand of the double-stranded target nucleic acid; and
(d) a second adapter nucleic acid sequence.
191 . The method of any one of claims 188 to 190 , wherein the second strand adapter-target nucleic acid sequence comprises in 5′ to 3′ order:
(a) a first adapter nucleic acid sequence comprising the first SDE;
(b) a second SMI domain;
(c) a second strand of the double-stranded target nucleic acid; and
(d) a second adapter nucleic acid sequence.
192 . The method of any one of claims 180 to 191 , wherein the consensus sequence for the first set of amplified products is compared to the consensus sequence for the second set of amplified products and a difference between the two consensus sequences is considered an artifact.
193 . A method of sequencing a double-stranded target nucleic acid comprising steps of:
(1) ligating a pair of adapter nucleic acid sequences of any one of claims 93 to 168 to at least one terminus of a double-stranded target nucleic acid molecule, thereby forming a double-stranded nucleic acid molecule comprising a first strand adapter-target nucleic acid sequence and a second strand adapter-target nucleic acid sequence; (2) amplifying the first strand adapter-target nucleic acid molecule, thereby producing a first set of amplified products comprising a plurality of first strand adapter-target nucleic acid molecules and a plurality of its complementary molecules; (3) amplifying the second strand adapter-target nucleic acid molecule, thereby producing a second set of amplified products comprising a plurality of second strand adapter-target nucleic acid molecules and a plurality of its complementary molecules; (4) sequencing the first set of amplified products, thereby obtaining a consensus sequence for the first set of amplified products; and (5) sequencing the second set of amplified products, thereby obtaining a consensus sequence for the second set of amplified products.
194 . The method of claim 193 , wherein the second set of amplified products is distinguishable from the first set of amplified products.
195 . The method of claim 193 or claim 194 , wherein amplification is performed by PCR.
196 . The method of claim 193 or claim 194 , wherein amplification is performed by multiple displacement amplification.
197 . The method of claim 193 or claim 194 , wherein amplification is performed by isothermal amplification.
198 . The method of claim 193 or claim 194 , further comprising after step (1), a step of contacting the double-stranded nucleic acid molecule with at least one enzyme that changes the at least one modified nucleotide to another chemical structure.
199 . The method of claim 198 , wherein the enzyme is a glycosylase.
200 . The method of claims 193 to 199 , wherein the pair of adapter nucleic acid sequences ligated to a first terminus of the double-stranded target nucleic acid molecule is identical to the pair of adapter nucleic acid sequences ligated to a second terminus of the double-stranded target nucleic acid molecule.
201 . The method of claims 193 to 199 , wherein the pair of adapter nucleic acid sequences ligated to a first terminus of the double-stranded target nucleic acid molecule is different from to the pair of adapter nucleic acid sequences ligated to a second terminus of the double-stranded target nucleic acid molecule.
202 . The method of claims 193 to 199 , wherein a pair of adapter nucleic acid sequences is ligated to a first terminus of a double-stranded target nucleic acid molecule and a primer corresponding to a portion of the DNA sequence of the target DNA molecule is utilized to amplify the DNA molecule.
203 . The method of any one of claims 193 to 200 , wherein the first strand adapter-target nucleic acid sequence comprises in 5′ to 3′ order:
(a) a first adapter nucleic acid sequence which comprises the at least one modified nucleotide or the at least one abasic site;
(b) a first strand of the double-stranded target nucleic acid; and
(c) a second adapter nucleic acid sequence.
204 . The method of any one of claims 193 to 203 , wherein the second strand adapter-target nucleic acid sequence comprises in 3′ to 5′ order:
(a) a first adapter nucleic acid sequence;
(b) a second strand of the double-stranded target nucleic acid; and
(c) a second adapter nucleic acid sequence.
205 . The method of claim 193 , wherein the pair of adapter nucleic acid sequences ligated to a first terminus of the double-stranded target nucleic acid molecule is different from the pair of adapter nucleic acid sequences ligated to a second terminus of the double-stranded target nucleic acid molecule.
206 . The method of claim 205 , wherein the pair of adapter nucleic acid sequences ligated to a first terminus of the double-stranded target nucleic acid molecule has a first SMI domain and the pair of adapter nucleic acid sequences ligated to a second terminus of the double-stranded target nucleic acid sequence has a second SMI domain;
wherein the first SMI domain is different from the second SMI domain.
207 . The method of claim 205 or claim 206 , wherein the first strand adapter-target nucleic acid sequence comprises in 5′ to 3′ order:
(a) a first adapter nucleic acid sequence comprising the at least one modified nucleotide or the at least one abasic site and the first SMI domain;
(b) a first strand of the double-stranded target nucleic acid; and
(c) a second adapter nucleic acid sequence comprising the second SMI domain.
208 . The method of claim 207 , wherein when the at least one modified nucleotide is 8-oxo-G, and the second adapter nucleic acid sequence includes a cytosine at a position corresponding to the 8-oxo-G.
209 . The method of any one of claims 205 to 208 , wherein the second strand adapter-target nucleic acid sequence comprises in 3′ to 5′ order:
(a) a first adapter nucleic acid sequence comprising the first SMI domain;
(b) a second strand of the double-stranded target nucleic acid; and
(c) a second adapter nucleic acid sequence comprising the second SMI domain.
210 . The method of claim 209 , wherein when the at least one modified nucleotide is 8-oxo-G, the second adapter nucleic acid sequence comprises a cytidine at a position corresponding to the 8-oxo-G.
211 . A method wherein distinguishable amplification products are obtained from each of the two strands of individual DNA molecules, and the consensus sequence for the first set of amplified products is compared to the consensus sequence for the second set of amplified products, wherein a difference between the two consensus sequences can be considered an artifact.
212 . The method of claim 211 , wherein amplified products are determined to have arisen from the same initial DNA molecule by virtue of sharing the same SMI sequence.
213 . The method of claim 211 , wherein amplified products are determined to have arisen from the same initial DNA molecule by virtue carrying distinct SMI sequences that are known to correspond to each other based upon a database produced at the time of and in conjunction with SMI adaptor library synthesis.
214 . The method of any one of claims 211 to 213 , wherein amplified products are determined to have arisen from distinct strands of the same initial double stranded DNA sequence via at least one nucleotide of sequence difference that was introduced by an SDE.
215 . A method wherein distinguishable amplification products are obtained from each of the two strands of individual DNA molecules, and the sequence obtained from an amplified product corresponding to one of the two initial DNA strands of a single DNA molecule is compared to an amplified product corresponding to the second of the two initial DNA strands, and a difference between the two sequences is considered an artifact.
216 . A method wherein indistinguishable amplification products are obtained from the two strands of an individual DNA molecule when the sequence obtained from an amplified product corresponding to one of the two initial DNA strands of a single DNA molecule is compared to an amplified product corresponding to the second of the two initial DNA strands and no difference between the two sequences is identified.
217 . The method of claim 215 or claim 216 , wherein amplified products are determined to have arisen from the same initial double stranded DNA molecule by virtue of sharing the same SMI sequence based upon database produced at the time of and in conjunction with SMI adaptor library synthesis.
218 . The method of claims 214 to 217 , wherein amplified products are determined to have arisen from distinct strands of the same initial double stranded DNA sequence via at least one nucleotide of sequence difference that was introduced by an SDE.
219 . The method of claims 214 to 218 , further comprising a step of single-molecule dilution following thermal or chemical melting of DNA duplexes into their component single-strands.
220 . The method of claim 219 , wherein the single-strands are diluted into multiple physically-separated reaction chambers such that the probability of the two originally paired strands sharing the same container is small.
221 . The method of claim 220 , wherein the physically-separated reaction chambers are selected from containers, tubes, wells, and at least a pair of non-communicating droplets.
222 . The method of claim 220 , wherein PCR amplification is carried out for each physically-separated reaction chamber, preferably using primers for each chamber carrying a different tag sequence.
223 . The method of claim 222 , wherein each tag sequence operates as an SDE.
224 . The method of claims 216 to 223 , wherein a series of paired sequences corresponding to the two strands of the same initial DNA are compared to one another, and at least one sequence from the series of products is selected as most likely to represent the correct sequence of the initial DNA molecule.
225 . The method of claim 224 , wherein the product selected as most likely to represent the correct sequence of the initial DNA molecule is selected at least in part due to having the smallest number of mismatches between the products obtained from the two DNA strands.
226 . The method of claim 224 or claim 225 , wherein the product selected as most likely to represent the correct sequence of the initial DNA molecule is selected at least in part due to having the smallest number of mismatches relative to the reference sequence.
227 . The method of any one of claims 203 to 210 , wherein during the amplification of step (2) or step (3), the at least one abasic site is converted upon amplification into a thymidine in the corresponding amplified product, resulting in introduction of an SDE.
228 . The method of claim 193 , wherein during the amplification of step (2) or step (3), the at least one modified nucleotide site encodes an adenosine in the corresponding amplified product.
229 . A composition comprising at least two pairs of adapter nucleic acid sequences, wherein a first pair of adapter nucleic acid sequences comprises:
a primer binding domain, a strand defining element (SDE), and a ligation domain;
wherein a second pair of adapter nucleic acid sequences comprises:
a primer binding domain,
a single molecule identifier (SMI) domain, and
a ligation domain.
230 . A double-stranded complexed nucleic acid comprising:
(1) a first pair of adapter nucleic acid sequences comprising:
a primer binding domain, and
an SDE, and
(2) a double-stranded target nucleic acid; and (3) a second pair of adapter nucleic acid sequences comprising:
a primer binding domain, and
a single molecule identifier (SMI) domain
wherein the first pair of adapter nucleic acid molecules is ligated to a first terminus of the double-stranded target nucleic acid molecule and the second pair of adapter nucleic acid molecules is ligated to a second terminus of the double-stranded target nucleic acid molecule.
231 . The double stranded complexed nucleic acid of claim 230 , wherein the first pair of adapter nucleic acid sequences and/or the second pair of adapter nucleic acid sequences further comprises a ligation domain.
232 . A pair of adapter nucleic acid sequences for use in sequencing a double-stranded target nucleic acid molecule, comprising a first adapter nucleic acid sequence and a second adapter nucleic acid sequence, wherein each adapter nucleic acid sequence comprises:
a primer binding domain, an SDE, a ligation domain; wherein the SDE of the first adapter nucleic acid sequence is at least partially non-complementary to the SDE of the second adapter nucleic acid sequence.
233 . A double-stranded circular nucleic acid comprising a pair of adapter nucleic acid molecules of any one of claims 1 to 80 ligated to a first terminus of a double-stranded target nucleic acid molecule and ligated to a second a second terminus of the double-stranded target nucleic acid molecule.
234 . A double-stranded circular nucleic acid comprising a pair of adapter nucleic acid molecules of any one of claims 93 to 168 ligated to a first terminus of a double-stranded target nucleic acid molecule and ligated to a second a second terminus of the double-stranded target nucleic acid molecule.
235 . A double-stranded circular nucleic acid comprising a pair of adapter nucleic acid molecules of any one of claims 1 to 80 ligated to a first terminus of a double-stranded target nucleic acid molecule and an annealed pair of primer binding domains ligated to a second terminus of the double-stranded target nucleic acid molecule;
wherein the annealed pair of primer binding domains is ligated to the pair of adapter nucleic acid molecules.
236 . A double-stranded circular nucleic acid comprising a pair of adapter nucleic acid molecules of any one of claims 93 to 168 ligated to a first terminus of a double-stranded target nucleic acid molecule and an annealed pair of primer binding domains ligated to a second terminus of the double-stranded target nucleic acid molecule;
wherein the annealed pair of primer binding domains is ligated to the pair of adapter nucleic acid molecules.
237 . A double-stranded complexed nucleic acid comprising:
(1) a pair of adapter nucleic acid sequences comprising:
a primer binding domain,
a strand defining element (SDE), and
a single molecule identifier (SMI) domain;
(2) a double-stranded target nucleic acid; and (3) an annealed pair primer binding domains;
wherein the pair of adapter nucleic acid molecules is ligated to a first terminus of the double-stranded target nucleic acid molecule and the annealed pair primer binding domains is ligated to a second terminus of the double-stranded target nucleic acid molecule.
238 . The double stranded complexed nucleic acid of claim 237 , wherein the pair of adapter nucleic acid sequences and/or the annealed pair primer binding domains further comprises a ligation domain.Join the waitlist — get patent alerts
Track US2022267841A1 — get alerts on status changes and closely related new filings.
We store only your email — no account needed. See our privacy policy.