Pacific Biosciences of California, Inc.

Leland Stanford Junior University

Pacific Biosciense of California, Inc.

Benjamin A Flusberg

Stephen Wilson Turner

Jonas Korlach

Jon M Sorenson

Kevin Travers

Jeremiah Hanes

Cheryl Heiner

Jeffrey Wegener

Patrick Marks

Mark Jacob Schnitzer

Dale R Webster

Robin A Emig

John Lyle

Lei Jia

Austin B Tomaney

Eric Cocker

Juergen Claus Jung

Eric Schadt

Mark Chaisson

Andrey Kislyuk

Jessica Lee

John S Eid

Adrian Nielsen Fehr

Lei Sun

Tyson A Clark

Igor D Vilfan

Joseph Puglisi

Robert Sebra

Daniel Bernardo Roitman

Keith P Bjornson

Paul Hardenbol

Mathieu Foquet

Fred C Christians

Donald M Gray

Jeremy Gray

Geoff Otto

Kenneth Mark Maxham

Tony H Ko

Stephen P Dudek

Hans Callebaut

Steven Lin

Bidhan P Chaudhuri

Ravi Dalal

Erik Paul Anderson

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

The invention relates to methods for nanopore sequencing. A substrate such as a semiconductor is provided having a nanopore or an array of nanopores connecting an upper solution with a lower solution. A polymerase-nucleic acid complex comprising a strand displacing DNA polymerase and a circular nucleic acid template and components required for DNA synthesis are provided. The polymerase produces a nascent DNA strand complementary to the circular nucleic acid template and this nascent strand is translocated through the nanopore while it is being produced by a polymerase-nucleic acid complex comprising a strand displacing DNA polymerase and a circular nucleic acid template and providing the components required for DNA synthesis whereby the polymerase produces a nascent DNA strand which is translocated through the nanopore while the nascent DNA strand is being produced. The sequence of the nascent DNA strand is determined while it translates through the pore using the measured current over time. The polymerase proceeds around the circular template and continues to produce the nascent DNA strand such that a sequence in the nascent strand corresponding a sequence in the circular template is determined more than once. Determining the sequence of corresponding to the circular template more than once can provide for greater accuracy.

Method of sequencing multiple copies of a sequence in a circular template

Identifying organisms in a sample using sequencing kinetic signatures

The invention relates to devices and methods for nanopore sequencing. The invention provides devices having an array of nanopores. The devices comprise an upper fluidic region in fluidic contact with the nanopores. The upper fluidic region comprises and upper electrode. Below each nanopore, and in fluidic contact with each nanopore is a discrete fluidic region. There is no direct fluidic contact between these discrete fluidic regions. The devices have an advantage of lower levels of electrical cross-talk between nanopores in the array for improved nucleic acid sequencing.

Nanopore sequencing using n-mers

The invention relates to devices and methods for nanopore sequencing. Methods for controlling translocation of through the nanopore are disclosed. The rate of transport of the template nucleic acids through the nanopore can be controlled using a translocating enzyme having two slow, or kinetically observable kinetic steps. The translocating enzyme and reaction conditions can be selected such that the translocating enzyme exhibits two kinetic steps wherein each of the kinetic steps has a rate constant, and the ratio of the rate constants of the kinetic steps is from 5:1 to 1:5. The invention also provides for using the signals from n-mers to provide sequence information, for example where the system has less than single base resolution. The invention includes arrays of nanopores having incorporated electronic circuits, for example, in CMOS.

Control of enzyme translocation in nanopore sequencing

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

Methods, compositions and arrays for non-random loading of single analyte molecules into array structures are provided. For example, methods are presented for providing a surface comprising the plurality of array regions by exposing the surface to a solution comprising polymerase enzymes where each polymerase enzyme is bound to a binding structure having several functional moieties. The functional moieties of the binding structure react with the binding elements on the array regions such that the functional moieties on the binding structure react with other available binding sites in an array region, preventing other polymerase-binding structures from loading, and resulting in a single polymerase molecule bound to each of these regions.

Single molecule loading methods and compositions

The invention relates to devices and methods for nanopore sequencing. The invention provides for using the signals from N monomeric units (n-mers) within the nanopore to provide sequence information. The invention includes substrates having arrays of nanopores with incorporated electronic circuits, for example, in semiconductor substrates. In some cases, the arrays of nanopores reside in discrete reservoirs.

Nanopore sequencing using N-mers

The present invention is generally directed to compositions, methods, and systems for performing single-molecule, real-time analysis of a variety of different biological reactions, and for determining various characteristics of the different biological reactions. The ability to analyze such reactions provides an opportunity to study those reactions as well as to potentially identify factors and/or approaches for impacting such reactions, e.g., to stimulate, enhance, or inhibit such reactions.

Real-time analytical methods and systems

The invention relates to devices and methods for nanopore sequencing. Methods for controlling translocation of through the nanopore are disclosed. The rate of transport of the template nucleic acids through the nanopore can be controlled using a translocating enzyme having two slow kinetic steps. The translocating enzyme and reaction conditions can be selected such that the translocating enzyme exhibits two kinetic steps wherein each of the kinetic steps has a rate constant, and the ratio of the rate constants of the kinetic steps is from 10:1 to 1:10. The invention also provides for using the signals from n-mers to provide sequence information, for example where the system has less than single base resolution. The invention includes arrays of nanopores having incorporated electronic circuits, for example, in CMOS.

Analysis of live beings is facilitated. According to an example embodiment of the present invention, a light-directing arrangement such as an endoscope is mounted to a live being. Optics in the light-directing arrangement are implemented to pass source light (e.g., laser excitation light) into the live being, and to pass light from the live being for detection thereof. The light from the live being may include, for example, photons emitted in response to the laser excitation light (i.e., fluoresced). The detected light is then used to detect a characteristic of the live being.

Live being optical analysis system and approach

The invention relates to devices and methods for nanopore sequencing. The invention provides for using the signals from n-mers to provide sequence information, for example where the system has less than single base resolution. The invention includes arrays of nanopores having incorporated electronic circuits, for example, in CMOS. In some cases, the arrays of nanopores comprise resistive openings for isolating the electronic signals for improved sequencing. Methods for controlling translocation of through the nanopore are disclosed.

Micro-optical imaging is facilitated. According to an example embodiment, a micro-optical probe arrangement includes a GRIN-type lens probe to direct light to and from a specimen. Compensation optics tailored to the probe and aberrations introduced by the lens are located in a light path through the lens, and compensate for the introduced aberrations. A light detector detects light from the specimen, as facilitated by the compensation optics, and generates data characterizing an image of the specimen.

Microendoscopy with corrective optics

Methods for identifying nucleic acid modifications

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Palo Alto, CA, United States of America

Benjamin A Flusberg