Abstract: The present disclosure provides methods and compositions for enhancing the processivity of a polymerase in catalyzing template-dependent DNA synthesis in high concentrations of salt. Also disclosed are methods and compositions for enhancing the assembly of polymerase-template complex compatible with active DNA synthesis in the presence of low levels of nucleotides and at a high temperature, such as temperatures at or near the melting temperature of the polymerase.

Abstract: The present disclosure is directed to compositions and methods for deriving a plurality of kinetics parameters (240) for at least two different enzyme variants in a multiplex manner using nanopore-based sequencing. In some embodiments, the systems and methods may be used to screen different nanopore variants, or different combinations of both nanopore variants and enzyme variants.

Abstract: Described herein is a variant pol6 polymerase having improved thermostability. The disclosed polymerases each contain one or more substitutions relative to SEQ ID NO: 1 selected from the group consisting of G12W/Y/F, K114W/F/I, L117L/F/P, N194F/W/V, M232W/G/R, G313E/Q/L, A451F/W/Y, K490W/F/Y, Q565Y/I/V, Q590P/V/Y, and D681G/N/H. Additionally, the present specification discloses substitutions that can reduce stuttering in such polymerases, the substitutions selected from the group consisting of N298L, L538R, P542A, I570H/T/W/R/N/G, N574L, E633W/F, S636F, E639K, and K655G.

Abstract: Disclosed is a novel structure of a nucleic acid template and the method of making and using the structure. The structure consists of a double-stranded circle with a single-stranded gap. The circular gapped structure includes an extendable end from which copying or sequencing can be initiated.

Abstract: The present disclosure relates to tagged nucleoside-5?-oligophosphates having a positively charged polymer tag structure and components thereof. Such nucleoside-5?-oligophosphates are useful, for example, in nanopore-based sequencing-by-synthesis applications. Also disclosed herein are nanopore constructs engineered to have additional negatively-charged moieties in the channel of the nanopore. Such nanopores can be useful, for example, for providing a repellant force against template and/or primer nucleic acids inserting into the pore during a nucleic sequence-by-synthesis process. The tagged nucleoside-5?-oligophosphates and nanopores disclosed herein can be used together to provide nanopore-based nucleic acid sequencing-by-synthesis systems and processes having reduced background tag levels and improved throughput.

Abstract: Disclosed are methods for isolating polymerase complexes from a mixture of polymerase complex components. The polymerase complexes can comprise a nanopore to provide isolated nanopore sequencing complexes. The methods relate to the positive and negative isolation of the polymerase complexes and/or nanopore sequencing complexes. Also disclosed is a nucleic acid adaptor for isolating active polymerase complexes, polymerase complexes comprising the nucleic acid adaptor, and methods for isolating active polymerase complexes using the nucleic acid adaptor.

Abstract: Described herein is a variant pol6 polymerase having at least one mutation selected from H223, N224, Y225, H227, I295, Y342, T343, I357, S360, L361, I363, S365, S366, Y367, P368, D417, E475, Y476, F478, K518, H527, T529, M531, N535, G539, P542, N545, Q546, A547, L549, 1550, N552, G553, F558, A596, G603, A610, V615, Y622, C623, D624, I628, Y629, R632, N635, M641, A643, I644, T647, I648, T651, I652, K655, W656, D657, V658, H660, F662, L690 and combinations thereof.

Abstract: Disclosed are methods for isolating polymerase complexes from a mixture of polymerase complex components. The polymerase complexes can comprise a nanopore to provide isolated nanopore sequencing complexes. The methods relate to the positive and negative isolation of the polymerase complexes and/or nanopore sequencing complexes. Also disclosed is a nucleic acid adaptor for isolating active polymerase complexes, polymerase complexes comprising the nucleic acid adaptor, and methods for isolating active polymerase complexes using the nucleic acid adaptor.

Abstract: Described herein are polymerase variants that are exonuclease deficient. Some variants retain the strand displacement capability comparable to the wild-type or parental polymerase. Some variants have a strand displacement capability that is improved relative to the wild-type or parental polymerase. The variants may have an extension rate that is greater than the wild-type or parental polymerase. The variants may have a waiting time that is less than the wild-type or parental polymerase.

Abstract: This application discloses compositions comprising primer compounds that reduce or block deleterious threading into a nanopore of nucleic acid strands displaced by a nanopore-linked polymerase, for example during the use of a nanopore device for nucleic acid sequencing. Also disclosed are methods for using the compositions to reduce deleterious threading events during nanopore-based nucleic acid detection techniques, such as nanopore sequencing.

Abstract: Disclosed are methods for isolating polymerase complexes from a mixture of polymerase complex components. The polymerase complexes can comprise a nanopore to provide isolated nanopore sequencing complexes. The methods relate to the positive and negative isolation of the polymerase complexes and/or nanopore sequencing complexes. Also disclosed is a nucleic acid adaptor for isolating active polymerase complexes, polymerase complexes comprising the nucleic acid adaptor, and methods for isolating active polymerase complexes using the nucleic acid adaptor.

Abstract: The present disclosure relates to tagged multi-nucleotide compounds, which comprise a single tag moiety covalently linked to a plurality of nucleoside-5?-oligophosphate moieties. As disclosed herein, these tagged multi-nucleotide compounds have improved characteristics as polymerase substrates and can be used in a range of nucleic acid detection and sequencing methods, including nanopore sequencing-by-synthesis.

Abstract: The present disclosure provides methods and compositions for enhancing the processivity of a polymerase in catalyzing template-dependent DNA synthesis in high concentrations of salt. Also disclosed are methods and compositions for enhancing the assembly of polymerase-template complex compatible with active DNA synthesis in the presence of low levels of nucleotides and at a high temperature, such as temperatures at or near the melting temperature of the polymerase.

Abstract: Described herein is a variant pol6 polymerase having at least one mutation selected from H223, N224, Y225, H227, I295, Y342, T343, I357, S360, L361, I363, S365Q, S366, Y367, P368, D417, E475, Y476, F478, K518, H527, T529, M531, N535, G539, P542, N545, Q546, A547, L549, I550, N552, G553, F558, A596, G603, A610, V615, Y622, C623, D624, I628, Y629, R632, N635, M641, A643, I644, T647, I648, T651, I652, K655, W656, D657, V658, H660, F662, L690 and combinations thereof.

Abstract: Described herein is a variant pol6 polymerase having at least one mutation selected from H223, N224, Y225, H227, I295, Y342, T343, I357, S360, L361, I363, S365, S366, Y367, P368, D417, E475, Y476, F478, K518, H527, T529, M531, N535, G539, P542, N545, Q546, A547, L549, 1550, N552, G553, F558, A596, G603, A610, V615, Y622, C623, D624,1628, Y629, R632, N635, M641, A643, 1644, T647, 1648, T651, 1652, K655, W656, D657, V658, H660, F662, L690 and combinations thereof.

Abstract: The present disclosure provides methods and compositions for enhancing the processivity of a polymerase in catalyzing template-dependent DNA synthesis in high concentrations of salt. Also disclosed are methods and compositions for enhancing the assembly of polymerase-template complex compatible with active DNA synthesis in the presence of low levels of nucleotides and at a high temperature, such as temperatures at or near the melting temperature of the polymerase.

Abstract: Described herein are polymerase variants that are exonuclease deficient. Some variants retain the strand displacement capability comparable to the wild-type or parental polymerase. Some variants have a strand displacement capability that is improved relative to the wild-type or parental polymerase. The variants may have an extension rate that is greater than the wild-type or parental polymerase. The variants may have a waiting time that is less than the wild-type or parental polymerase.

Abstract: The present disclosure relates to tagged multi-nucleotide compounds, which comprise a single tag moiety covalently linked to a plurality of nucleoside-5?-oligophosphate moieties. As disclosed herein, these tagged multi-nucleotide compounds have improved characteristics as polymerase substrates and can be used in a range of nucleic acid detection and sequencing methods, including nanopore sequencing-by-synthesis.

Abstract: Described herein is a variant pol6 polymerase having at least one mutation selected from H223, N224, Y225, H227, I295, Y342, T343, I357, S360, L361, I363, S365, S366, Y367, P368, D417, E475, Y476, F478, K518, H527, T529, M531, N535, G539, P542, N545, Q546, A547, L549, I550, N552, G553, F558, A596, G603, A610, V615, Y622, C623, D624, I628, Y629, R632, N635, M641, A643, I644, T647, I648, T651, I652, K655, W656, D657, V658, H660, F662, L690 and combinations thereof.

Abstract: Described herein is a variant pol6 polymerase having at least one mutation selected from H223, N224, Y225, H227, I295, Y342, T343, I357, S360, L361, I363, S365Q, S366, Y367, P368, D417, E475, Y476, F478, K518, H527, T529, M531, N535, G539, P542, N545, Q546, A547, L549, I550, N552, G553, F558, A596, G603, A610, V615, Y622, C623, D624, I628, Y629, R632, N635, M641, A643, I644, T647, I648, T651, I652, K655, W656, D657, V658, H660, F662, L690 and combinations thereof.