Roche Sequencing Solutions, Inc.

Roche Molecular Systems, Inc.

Genia Technologies, Inc.

Harvard College

Roche Diagnostics Gmbh

F. Hoffmann-La Roche Ag

Aruna Ayer

George M Church

Frank Bergmann

Christoph Seidel

Ilya Lederman

Eileen Thai

Arkadiusz Bibillo

Charles Wayan Schwab

Colin Alexander McGaw

Cleoma Arnold

Preethi Sarvabhowman

Tyler O'Brien Shultz

Shawn Lee Suko

Mara Boenitz-Dulat

Peter Crisalli

Barbara Eckert

Bigna Woersdoerfer

David Daniel Wunderlich

Dhruti Vasudev Dalal

Randall W Davis

Cynthia Ann Cech

Timothy Kellogg Craig

Helen Franklin

Natalie B Chechelski Johnston

Dmitriy Gremyachinskiy

Charles Schwab

Meng Taing

Marshall Winston Porter

Hannes Kuchelmeister

Omid Khakshoor

Kirti Dhiman

Joshua N Mabry

Evan Amato

Mirkó Palla

Francois Pepin

Sukanya Srinivasa Rao Arun Punthambaker

Peter B Stranges

Tyler O'Brien Schultz

Cleoma Renetta Arnold

Shawn Suko

Rongxin Nie

Cynthia Cech

Colin Mcgaw

Tyler Shultz

Seong-Ho Shin

Lars Hillringhaus

The present disclosure is directed to compositions and methods for deriving a plurality of kinetics parameters () 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.

Enzyme screening methods

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.

Purification of polymerase complexes

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.

Polymerase variants and uses thereof

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.

Exonuclease deficient polymerases

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.

Polymerase-template complexes

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.

Tagged multi-nucleotides useful for nucleic acid sequencing

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.

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.

Polymerase variants

The present disclosure provides variant Pol6 polymerase polypeptides, compositions comprising the Pol6 variant polypeptides, and methods for using the variant Pol6 polypeptides for determining the sequencing of nucleic acids, for example, by nanopore sequencing. The variant Pol6 polymerases possess decreased rates of dissociation of template from the polymerase-template complex, which result in increased processivity relative to the parental Pol6 polypeptides from which they are derived.

Pol6 polymerase variants

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, 1644, T647, I648, T651, I652, K655, W656, D657, V658, H660, F662, L690 and combinations thereof.

Growing community of inventors

Santa Clara, CA, United States of America

Aruna Ayer