Pacific Biosciences of California, Inc.

Levitasbio, Inc.

Kevin Travers

Cheryl Heiner

Stephen Wilson Turner

Jonas Korlach

Tyson A Clark

Jon M Sorenson

Geoff Otto

Congcong Ma

Benjamin A Flusberg

Keith P Bjornson

Patrick Marks

Thang Pham

Arunashree Bhamidipati

Eric Olivares

Erik Miller

Jeremiah Hanes

Robin A Emig

Dale R Webster

John Lyle

Fred C Christians

John S Eid

Lei Jia

Arek Bibillo

Austin B Tomaney

Kenneth Mark Maxham

Andrey Kislyuk

Jessica Lee

Pranav Patel

Jeffrey Wegener

Donald M Gray

Adrian Nielsen Fehr

Jeremy Gray

Igor D Vilfan

Yu-Chih Tsai

Joseph Puglisi

Mark Chaisson

Lesley Suzanne Weaver

Aj Chmura

Theodorus Evan De Groot

Geoffrey Facer

Pham Thang

Methods, compositions, and systems are provided that allow for reliable sequencing of the initial sequence region of a sequence of interest. The methods of the invention allow for more reliable barcoding of subpopulations of nucleic acids to be sequenced.

Delaying real-time sequencing

The present invention relates generally to systems, materials, and methods utilizing magnetic levitation to affect separation and/or concentration of particulate containing samples, such as cells or biomolecules, within a medium. In some embodiments, the methods are used for the separation of a mixture of live cells and dead cells, and to isolate particle depleted medium.

Particle separator system, materials, and methods of use

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.

Classification of nucleic acid templates

Compositions, methods and systems are provided for isolating nucleic acids. A polymerase-nucleic acid complex can be formed by mixing a polymerase enzyme comprising strand displacement activity and a mixture of double stranded nucleic acids. Nucleic acid synthesis can then be initiated by the polymerase enzyme to produce a nascent strand complementary to the first strand, thereby displacing a portion of the second strand. After halting or reducing the rate of nucleic acid synthesis, a hybridizing a hook oligonucleotide can be used hybridize to the nucleic acid through a capture region on the hook oligonucleotide that is complementary to the displaced portion of the second strand. The nucleic acid can then be isolated from the mixture of nucleic acids using the hook oligonucleotide.

Methods for isolating nucleic acids

Methods and systems for single molecule sequencing using concatemers of copies of sense and antisense strands. Concatemers are provided, for example, by carrying out rolling circle amplification on a circular molecule having sense and antisense regions to produce repeated copies of the sense and antisense regions connected by linking regions. The circular molecules can be produced by ligating hairpin adapters to each end of a double-stranded nucleic acid having a sense and antisense strand. The ligations can be carried out, for example using blunt end ligation. In some cases, a single molecule consensus sequence for a single template molecule is obtained. A single read from each template molecule can be obtained by comparing the sequence information of the sense and antisense regions.

Sequencing using concatemers of copies of sense and antisense strands

Compositions, methods and systems are provided for the loading of extended polymerase-nucleic acid complexes onto substrates. Primed polymerase-template complex comprising a polymerase enzyme and a circular nucleic acid template comprising a double stranded region connected at each end by a single-stranded hairpin region are provides in which the circular nucleic acid template comprises at least one reversible pause point. Nucleic acid synthesis is carried out such that a nascent strand is synthesized up to the reversible stop point producing an extended polymerase-template complex. The extended polymerase-template complex is then attached to a substrate. Such attached complexes can be used for single molecule sequencing in which the nucleic acid synthesis is re-initiated such that nucleic acid synthesis continues past the reversible stop point.

Loading extended polymerase-nucleic acid complexes

Identifying organisms in a sample using sequencing kinetic signatures

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

Compositions, methods and systems are provided for isolating DNA having a modified or unnatural base. Circular DNA fragments, each comprising a double stranded DNA central region and single stranded regions on the ends of the double stranded regions, are obtained. Some of the fragments have one or more modified or unnatural base. The DNA fragments are treated with a primer and a polymerase such that the polymerase extends the primer to copy at least one of the strand of the double stranded region. This results in rendering the other strand single stranded. A binding protein or antibody that is specific to the modified or unnatural base is then used to isolate strands containing the modified or unnatural bases. Methods for loading such complexes onto substrates and for single molecule sequencing of such complexes are also provided.

Isolation of polymerase-nucleic acid complexes

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. Methods are provided that include linking forward and reverse strands of a double stranded segment to form circular template molecules, and obtaining sequence data from the circular template molecule to identify modified bases, for example including the use of bisulfite treatment. 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.

Methods of sequencing and identifying the position of a modified base in a nucleic acid

Methods, devices, and systems for performing intermittent detection during analytical reactions are provided. Such methods facilitate collection of reaction data from disparate reaction times. Further, such methods are useful for reducing photo-induced damage of one or more reactants in an illuminated analytical reaction at a given reaction time. In preferred embodiments, the reaction mixture is subjected to at least one illuminated and non-illuminated period and allowed to proceed such that the time in which the reaction mixture is illuminated is less than a photo-induced damage threshold period.

Nucleic acid sequence analysis

Computer systems, computer readable media, and computer methods for obtaining, calling, and assembling nucleic acid sequences are presented. In some aspects the invention includes the sequencing of template constructs that comprise double stranded portions in partially contiguous constructs, to provide for single molecule consensus sequence determination through one or both of sequencing sense and antisense strands in the same molecule.

Methods and systems for obtaining a single molecule consensus sequence

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Menlo Park, CA, United States of America

Kevin Travers