Becton Dickinson and Company Limited

Cellular Research, Inc.

Affymetrix, Inc.

Incyte Genomics, Inc.

Incyte Corporation

Incyte Pharmaceuticals, Inc.

Glenn K Fu

Stephen P A Fodor

Geoffrey Richard Facer

Christina Fan

Julie Wilhelmy

Eleen Shum

Pei-Hua Wang

Michael H Shapero

Craig Betts

Keith W Jones

Laura L Stuve

Robert G Kuimelis

Jing Hu

David Stern

Walter H Lee

Andrew Sparks

Roger Rudoff

George T Wu

Steven Starnes

Glenn H Mcgall

Glenn H McGall

Gretchen Yinbon Lam

Jue Fan

Lisha Deng

Irene Ni

Jennifer Tsai

Jonathan T Wang

Stephen Pa Fodor

Methods are provided for multiplexed amplification of selected targets and analysis of the amplified targets. In preferred aspects the amplification and analysis take place on the same solid support and preferably in a localized area such as a bead or a feature of an array. In preferred aspects the analysis is a determination of sequence at one or more locations in the amplified target. The methods may be used for genotyping, sequencing and analysis of copy number.

Locus specific amplification using array probes

Compositions, methods and kits are disclosed for high-sensitivity single molecule digital counting by the stochastic labeling of a collection of identical molecules by attachment of a diverse set of labels. Each copy of a molecule randomly chooses from a non-depleting reservoir of diverse labels. Detection may be by a variety of methods including hybridization based or sequencing. Molecules that would otherwise be identical in information content can be labeled to create a separately detectable product that is unique or approximately unique in a collection. This stochastic transformation relaxes the problem of counting molecules from one of locating and identifying identical molecules to a series of binary digital questions detecting whether preprogrammed labels are present. The methods may be used, for example, to estimate the number of separate molecules of a given type or types within a sample.

Digital counting of individual molecules by stochastic attachment of diverse labels

This disclosure provides methods and compositions for removing one or more high abundance species from a plurality of nucleic acid molecules. In some embodiments, the methods and compositions can be used for normalizing nucleic acid libraries. In some embodiments, molecular labels are used in conjunction with the methods and compositions disclosed herein to improve sequencing efficiency.

Normalization of nucleic acid libraries

The disclosure provides for methods, compositions, and kits for multiplex nucleic acid analysis of single cells. The methods, compositions and systems may be used for massively parallel single cell sequencing. The methods, compositions and systems may be used to analyze thousands of cells concurrently. The thousands of cells may comprise a mixed population of cells (e.g., cells of different types or subtypes, different sizes).

Massively parallel single cell analysis

Methods, kits and systems are disclosed for analyzing one or more molecules in a sample. Analyzing the one or more molecules may comprise quantitation of the one or more molecules. Individual molecules may quantitated by PCR, arrays, beads, emulsions, droplets, or sequencing. Quantitation of individual molecules may further comprise stochastic labeling of the one or more molecules with a plurality of oligonucleotide tags to produce one or more stochastically labeled molecules. The methods may further comprise amplifying, sequencing, detecting, and/or quantifying the stochastically labeled molecules. The molecules may be DNA, RNA and/or proteins.

Compositions and kits for molecular counting

The invention relates to methods for enzymatic, genotyping of polymorphisms on solid supports. In some aspects, the methods include ligation of allele or base specific 5' interrogation probes to an array probe. The array probe is labeled by ligation of the interrogation probe. Ligation is dependent on the identity of the base immediately adjacent to the end of the array probe. In other aspects array bound probes are labeled by template dependent extension.

Enzymatic methods for genotyping on arrays

The present disclosure provide compositions, methods and kits for generating a set of combinatorial barcodes, and uses thereof for barcoding samples such as single cells or genomic DNA fragments. Some embodiments disclosed herein provide compositions comprising a set of component barcodes for producing a set of combinatorial barcodes. The set of component barcodes can comprise, for example, n&#xd7;m unique component barcodes, wherein n and m are integers, each of the component barcodes comprises: one of n unique barcode subunit sequences; and one or two linker sequences or the complements thereof, wherein the component barcodes are configured to connect to each other through the one or two linker sequences or the complements thereof to produce a set of combinatorial barcodes.

Methods and compositions for combinatorial barcoding

Disclosed herein are methods and systems for determining the numbers of targets. In some embodiments, the method comprise: stochastically barcoding targets using stochastic barcodes; obtaining sequencing data; for one or more of the targets: counting the number of molecular labels with distinct sequences associated with the target in the sequencing data; identifying clusters of molecular labels of the target using directional adjacency; collapsing the sequencing data using the clusters of molecular labels of the target identified; and estimating the number of the target.

Molecular label counting adjustment methods

The disclosure provides for methods, compositions, systems, devices, and kits for whole transcriptome amplification using stochastic barcodes.

Methods and compositions for whole transcriptome amplification

The disclosure provides for methods, compositions, and kits for normalizing nucleic acid libraries, for example sequencing libraries.

Methods and compositions for library normalization

Disclosed herein include methods and compositions for selectively amplifying and/or extending nucleic acid target molecules in a sample. The methods and compositions can, for example, reduce the amplification and/or extension of undesirable nucleic acid species in the sample, and/or allow selective removal of undesirable nucleic acid species in the sample.

Selective amplification using blocking oligonucleotides

Growing community of inventors

Menlo Park, CA, United States of America

Glenn K Fu