Roche Sequencing Solutions, Inc.

Genia Technologies, Inc.

Other

Columbia University

Life Technologies Corporation

C. R. Bard, Inc.

Roche Molecular Systems, Inc.

Ave Connaught

Roche Diagnostics Gmbh

F. Hoffmann-la Roche Ag

Randall W Davis

Roger Chen

Roger J A Chen

Cynthia Ann Cech

Arkadiusz Bibillo

Edward Shian Liu

Andrew Trans

Eric Takeshi Harada

Anne Aguirre

James Pollard

Gregory R Whittaker

Jingyue Ju

Aruna Ayer

James E Lock

Timothy Kellogg Craig

Peter Crisalli

Helen Franklin

Daniel Korenblum

Gerry D Ouellette

George Bourne

George Duval

Maria S Wagner

Stanton B Perry

Dennis Goodine

Kirti Dhiman

Natalie B Chechelski Johnston

Joshua N Mabry

Hui Tian

Shiv R Kumar

Carl Williams Fuller

John S Foster

Timothy G Geiser

Robert A Yuan

Michael Dorwart

Janine Mok

Cynthia Cech

William A Berthiaume

Kevin Deierling

Markus Wallgren

Thomas Farrell

Kevin Treacy

A method for sequencing a nucleic acid molecule includes providing a sequencing cell having a nanopore in a membrane that resides over a well, a first electrode at a bottom of the well, a second electrode in a chamber above the membrane, and an electrolyte in the well and the chamber. The first electrode is configured to facilitate non-Faradaic conduction of ionic current and forms a capacitance with ions in the electrolyte. A first voltage signal is applied across the first and second electrodes, thereby creating a force that moves a nucleic acid molecule in the sequence cell through the nanopore. The first voltage signal increases to compensate for changes in the capacitance at the first electrode during application of the first voltage signal. The method further includes determining signal values measured during the first voltage signal, which correspond to one or more nucleotides in the nucleic acid molecule.

Nanopore voltage methods

This disclosure provides a biochip comprising a plurality of wells. The biochip includes a membrane that is disposed in or adjacent to an individual well of the plurality of wells. The membrane comprises a nanopore, and the individual well comprises an electrode that detects a signal upon ionic flow through the pore in response to a species passing through or adjacent to the nanopore. The electrode can be a non-sacrificial electrode. A lipid bilayer can be formed over the plurality of wells using a bubble.

Methods for forming lipid bilayers on biochips

A method of forming a nanopore in a lipid bilayer is disclosed. A nanopore forming solution is deposited over a lipid bilayer. The nanopore forming solution has a concentration level and a corresponding activity level of pore molecules such that nanopores are substantially not formed un-stimulated in the lipid bilayer. Formation of a nanopore in the lipid bilayer is initiated by applying an agitation stimulus level to the lipid bilayer. In some embodiments, the concentration level and the corresponding activity level of pore molecules are at levels such that less than 30 percent of a plurality of available lipid bilayers have nanopores formed un-stimulated therein.

Methods for forming a nanopore in a lipid bilayer

The present disclosure provides biochips and methods for making biochips. A biochip can comprise a nanopore in a membrane (e.g., lipid bilayer) adjacent or in proximity to an electrode. Methods are described for forming the membrane and inserting the nanopore into the membrane. The biochips and methods can be used for nucleic acid (e.g., DNA) sequencing. The present disclosure also describes methods for detecting, sorting, and binning molecules (e.g., proteins) using biochips.

Methods for creating bilayers for use with nanopore sensors

This disclosure provides systems and methods for molecular identification and polymer (e.g., nucleic acid) sequencing using nanopores. The polymer may be passed through or in proximity to the nanopore and various subunits of the polymer may affect the current flowing through the nanopore. The various subunits may be identified by measuring the current at a plurality of voltages applied across the nanopore and/or membrane. In some cases, the polymerization of tagged nucleotides presents tag molecules to the nanopore that can be identified by measuring the current at a plurality of voltages applied across the nanopore and/or membrane. Also provided herein are systems and methods for sequencing both the sense and anti-sense strand of a double stranded nucleic acid molecule with a nanopore and methods for using ribonucleic acid (RNA) speed bump molecules to slow the passage of a nucleic acid molecule through or in proximity to a nanopore.

Nanopore based molecular detection and sequencing

Provided are methods of nucleic acid sample preparation for analysis, including analysis using nanopore sequencing applications. In particular provided are methods that allow for the creation of circular and linear DNA samples that contain asymmetric ends to ligate different and desired adaptors to the end of the sample, methods to create single nucleic acid molecules that automatically form concatemers for higher sequencing throughput; methods to create multiple copies (temperature dependent, multi-primed, open-fold replication) of one original nucleic acid sample, or to create single nucleic acid molecules containing multiple copies of more than one original nucleic acid sample (concatemerized samples) for improved accuracy and throughput are described; and methods for improved modified base calling.

Nucleic acid sample preparation 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

The present invention relates to a method of using nanopores to obtain sequence information of sample DNAs in ss test DNAs. The method comprises using speed bumps to stall the ss test DNAs in the nanopores at random positions of the ss test DNAs to obtain sequence information of each and every nucleotides of the sample DNAs, and to construct the whole sequences of the sample DNAs. The present invention also relates to identification and/or isolation of test DNAs having desired sequence(s) using nanopore detectors facilitated by speed bump.

Nanopore-based single DNA molecule characterization, identification and isolation using speed bumps

This disclosure provides systems and methods for sequencing nucleic acids using nucleotide analogues and translocation of tags from incorporated nucleotide analogues through a nanopore. In aspects, this disclosure is related to compositions, methods, and systems for sequencing nucleic acids using tag molecules and detection of translocation through a nanopore of tags released from incorporation of the molecule.

Nucleic acid sequencing by nanopore detection of tag molecules

Sequencing adaptors and methods are provided for preparation of polynucleotides for sequencing. The sequencing adaptors contain a portion of a recognition sequence for a methyl-dependent endonuclease. Unwanted adaptor dimers that form during ligation of adaptors to target polynucleotides produce a complete restriction sequence and are cleaved by the endonuclease, followed by exonuclease digestion, thereby removing the dimers.

Methods for removal of adaptor dimers from nucleic acid sequencing preparations

Systems and methods of polynucleotide sequencing are provided. Systems and methods optimize control, speed, movement, and/or translocation of a sample (e.g., a polynucleotide) within, through, or at least partially through a nanopore or a type of protein or mutant protein in order to accumulate sufficient time and current blocking information to identify contiguous nucleotides or plurality of nucleotides in a single-stranded area of a polynucleotide.

Systems and methods for determining genetic data

Growing community of inventors

Santa Clara, CA, United States of America

Randall W Davis