Pacific Biosciences of California, Inc.

John Lyle

Andrei Fedorov

Jeremiah Hanes

Stephen Wilson Turner

David R Rank

Jonas Korlach

Stephen T Yue

Paul S Peluso

Geoff Otto

Gene W Shen

Daniel Bernardo Roitman

Lubomir Sebo

Yue Xu

Keith P Bjornson

Fred C Christians

Louis Brogley

Insil Park

Honey Osuna

Yuri Lapin

Sonya Clark

Frank Zheng

Jeffrey Wegener

Thang Pham

Lei Jia

David Kagen Hanzel

Jeffery Wegener

Arekadiusz Bibillo

Kevin Travers

Benjamin A Flusberg

Jon M Sorenson

Xiangxu Kong

Dale R Webster

Robert Joseph Weber

Jessica Lee

Ron L Cicero

Cheryl Heiner

Patrick Marks

Lei Sun

Arek Bibillo

Tyson A Clark

Austin B Tomaney

Igor D Vilfan

Ronald L Cicero

Paul Mitsis

Andrey Kislyuk

Joseph Puglisi

Paul M Lundquist

Molly Min He

Robin A Emig

Geoffrey Alan Otto

Stephen P Dudek

Devon Murphy

Duc Do

Khai Luong

Harold Ray Lee

Mark Chaisson

Ravi Dalal

Christopher Silk

Grace Lee

David K Hanzel

Protected fluorescent reagent compounds and their methods of synthesis are provided. The compounds are useful in various fluorescence-based analytical methods, including the analysis of highly multiplexed optical reactions in large numbers at high densities, such as single molecule real time nucleic acid sequencing reactions. The compounds contain fluorescent dye elements, that allow the compounds to be detected with high sensitivity at desirable wavelengths, binding elements, that allow the compounds to be recognized specifically by target biomolecules, and protective shield elements, that decrease undesirable contacts between the fluorescent dye elements and the bound target biomolecules and that therefore decrease photodamage of the bound target biomolecules by the fluorescent dye elements.

Protected fluorescent reagent compounds

Compositions that include polymerases with features for improving entry of nucleotide analogues into active site regions and for coordinating with the nucleotide analogues in the active site region are provided. Methods of making the polymerases and of using the polymerases in sequencing and DNA replication and amplification as well as kinetic models of polymerase activity and computer-implemented methods of using the models are also provided.

Polymerases for nucleotide analogue incorporation

Methods of producing substrates having selected active chemical regions by employing elements of the substrates in assisting the localization of active chemical groups in desired regions of the substrate. The methods may include optical, chemical and/or mechanical processes for the deposition, removal, activation and/or deactivation of chemical groups in selected regions of the substrate to provide selective active regions of the substrate.

Articles having localized molecules disposed thereon and methods of producing same

Multimeric protected fluorescent reagents and their methods of synthesis are provided. The reagents are useful in various fluorescence-based analytical methods, including the analysis of highly multiplexed optical reactions in large numbers at high densities, such as single molecule real time nucleic acid sequencing reactions. The reagents contain fluorescent dye elements, that allow the compounds to be detected with high sensitivity at desirable wavelengths, binding elements, that allow the compounds to be recognized specifically by target biomolecules, and protective shield elements, that decrease undesirable contacts between the fluorescent dye elements and the bound target biomolecules and that therefore decrease photodamage of the bound target biomolecules by the fluorescent dye elements. The reagents also contain coupling elements connect monomeric compounds into multimeric forms, thereby increasing brightness.

Multimeric protected fluorescent reagents

Compositions, kits, methods and systems for single molecule nucleotide sequencing comprising producing polymerase reactions having monovalent cations that control the median pulse width for incorporated nucleotides are disclosed. The levels of monovalent cations are used to control pulse width while allowing other sequencing parameters to remain within a desirable range.

Sequencing reactions with monovalent cations for pulse width control

Methods, compositions, and systems are provided for characterization of modified nucleic acids. In certain preferred embodiments, single molecule sequencing methods are provided for identification of modified nucleotides within nucleic acid sequences. Modifications detectable by the methods provided herein include chemically modified bases, enzymatically modified bases, abasic sites, non-natural bases, secondary structures, and agents bound to a template nucleic acid.

Identification of nucleic acid template-linked barcodes comprising nucleic acid modifications

Compositions, kits, methods and systems for single molecule nucleotide sequencing comprising producing polymerase reactions having lithium that control the median pulse width for incorporated nucleotides are disclosed. The levels of lithium are used to control pulse width while allowing other sequencing parameters to remain within a desirable range.

Methods for performing analytical reactions and compositions for use in such methods, where the methods have reduced signal levels deriving from non-specific adsorption of detected reagents to other components of the analytical method, e.g., other reagents, solid phase components, vessels, etc.

Methods and compositions for performing analytical operations

Compositions that include polymerases with features for improving entry of nucleotide analogs into active site regions and for coordinating with the nucleotide analogs in the active site region are provided. Methods of making the polymerases and of using the polymerases in sequencing and DNA replication and amplification as well as kinetic models of polymerase activity and computer-implemented methods of using the models are also provided.

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Fremont, CA, United States of America

John Lyle