Pacific Biosciences of California, Inc.

10x Genomics, Inc.

Life Technologies Corporation

Keith P Bjornson

Jeremiah Hanes

Satwik Kamtekar

Erik Miller

Fred C Christians

Arek Bibillo

Pranav Patel

Stephen Wilson Turner

Robin A Emig

Lubomir Sebo

Lei Jia

John Lyle

Jonas Korlach

Kevin Travers

Louis Brogley

Andrei Fedorov

Harold Ray Lee

Insil Park

Sonya Clark

Benjamin J Hindson

Molly Min He

Paul Hardenbol

Colleen Cutcliffe

Walter H Lee

Indira Wu

Paul William Wyatt

Cheryl Heiner

Stephen P Hendricks

Paul S Peluso

Lei Xi

Kamila Belhocine

Stephen T Yue

Gene W Shen

Mathieu Foquet

Arkadiusz Bibillo

Benjamin A Flusberg

Adrian Nielsen Fehr

Jon M Sorenson

Zahra Kamila Belhocine

Natasha Popovich

Hans Callebaut

Bidhan P Chaudhuri

Mark Chaisson

Steven Lin

Robert Sebra

Paul Wyatt

Leewin Chern

Zhong Wang

Kristofor Nyquist

Christopher Silk

John M Lyle

Compositions, methods, and systems are provided for fluorescent polymerase enzyme substrates comprising protein shields for improving enzyme photostability in single molecule real time sequencing. Fluorescent polymerase enzyme substrates of the invention have a protein shield between the fluorescent dye moieties and nucleotide moieties of the polymerase enzyme substrate. The polymerase enzyme substrates have a nucleotide component and a dye component, each attached to a protein. The attachments can be covalent. The protein can, for example, prevent the direct interaction of the fluorescent dye moiety with the enzyme when carrying out nucleotide synthesis, preventing photodamage to the enzyme. The polymerase enzyme substrates of the invention can have multiple dyes and multiple nucleotide moieties.

Fluorescent polymerase enzyme substrates having protein shields

This disclosure provides methods for preparing a sequencing library including the steps of providing a template nucleic acid sequence, dNTPs, dUTP, a primer, a polymerase, a dUTP excising enzyme, and a plurality of beads including oligonucleotide adapter sequence segments; amplifying the template nucleic acid with the polymerase, dNTPs, dUTP and random hexamer to provide a complementary nucleic acid sequence including occasional dUTPs; and excising the incorporated dUTPs with the dUTP excising enzyme to provide nicks in the complementary nucleic acid sequence to provide a sequencing library.

Processes and systems for preparation of nucleic acid sequencing libraries and libraries prepared using same

Sequencing methods, devices, and systems are described. Arrays of nanoscale electronic elements comprising two electrodes separated by an insulating layer are used to provide sequence information about a template nucleic acid in a polymerase-template complex bound proximate to the insulating region. A sequencing reaction mixture comprising nucleotide analogs having impedance labels is introduced to the array of nanoscale electronic elements under conditions of polymerase mediated nucleic acid synthesis. The time sequence of incorporation of nucleotide analogs is determined by identifying the types of labels of the nucleotide analogs that are incorporated into the growing strand using measured impedance.

Nucleic acid sequencing with nanoscale electrode pairs

This specification generally relates to non-radioactive methods of detecting nucleic acid polymerase activity and methods of detecting compounds that modulate nucleic acid polymerase activity. The activity may be measured in real-time using a real-time PCR instrument.

Polymerase assay with a FRET substrate

Provided are compositions comprising recombinant DNA polymerases that include amino acid substitutions, insertions, deletions, and/or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing. Such properties include increased resistance to photodamage, and can also include enhanced metal ion coordination, reduced exonuclease activity, reduced reaction rates at one or more steps of the polymerase kinetic cycle, decreased branching fraction, altered cofactor selectivity, increased yield, increased thermostability, increased accuracy, increased speed, increased readlength, and the like. Also provided are nucleic acids which encode the polymerases with the aforementioned phenotypes, as well as methods of using such polymerases to make a DNA or to sequence a DNA template.

Recombinant polymerases with increased phototolerance

The present invention provides methods, compositions, and systems for distributing molecules and complexes into reaction sites. In particular, the methods, compositions, and systems of the present invention result in an active loading of molecules and complexes into reaction sites with improved efficiency over loading by passive diffusion methods alone.

Methods and compositions for delivery of molecules and complexes to reaction sites

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

Multi-biotinylated reactants are provided which can be used in divalent complexes for various applications such as colocalization, labeling, immobilization, and purification. Methods for constructing, purifying, and using the bis-biotinylated reactants are also provided. In certain embodiments, two bis-biotinylated reactants are bound to a single streptavidin tetramer to provide a complex having a 1:1 stoichiometry with respect to the bis-biotinylated reactants.

Bis-biotinylation tags

The present invention provides methods, compositions, and systems for enriching compositions for polymerase enzyme complexes. In particular, the methods, compositions, and systems of the present invention remove free polymerases from the compositions using one or more purification steps, including protease treatment, thus enriching the compositions for polymerases complexed with a template nucleic acid.

Methods and compositions for enriching compositions for polymerase enzyme complexes

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Union City, CA, United States of America

Keith P Bjornson