The Broad Institute, Inc.

Dna Script Sas

Other

Massachusetts Institute of Technology

Ecole Superieure De Physique Et De Chimie Industrielles De La Ville De Paris

Harvard College

The General Hospital Corporation D/b/a Massachusetts General Hospital

Nicol, Robert

Steelman, Scott

Lintner, Robert, E.

Espci Innov Sas

Robert Nicol

David A Weitz

Scott Steelman

Thomas Ybert

Sylvain Gariel

Andrew David Griffiths

Jeffrey Jeddeloh

Xavier Godron

Adrian Horgan

Robert E Lintner

Niall J Lennon

Christopher A Voigt

Patrick Tabeling

Tarjei Mikkelsen

Alon Goren

Marie Leman

Baptiste Saudemont

Timothy V Kirk

Lauren Andrews

Xiaofeng Xin

Harris Nusbaum

Bradley E Bernstein

The invention is directed to methods for synthesizing oligonucleotides directly on biomolecules or cells living or fixed. In some embodiments, template-free enzymatic synthesis is implemented under biological conditions with successive cycles of (i) enzymatic addition of a 3'-O-blocked nucleoside triphosphate and (ii) enzymatic deblocking of the incorporated nucleotide to regenerate a free 3′ hydroxyl. The invention has applications in single-cell cDNA library construction and analysis.

Methods for extending polynucleotides

Disclosed is a method of generating a set of sequence-verified nucleic acid elements for the combinatorial construction of genetic elements. The method includes: providing a plurality of nucleic acid parts; assembling nucleic acid parts to form a one or more nucleic acid elements, wherein the nucleic acid elements include at least two sequences selected from the plurality of parts; and determining the sequence of the nucleic acid elements. Further disclosed is a pool of higher-order nucleic acid constructs or amplification products thereof, comprising one or more nucleic acid elements as well as kits including a pool of sequence-verified nucleic acid elements of claims and/or a pool of higher-order nucleic acid constructs; and a plurality of primers for retrieving one or more sequence-verified nucleic acid elements and/or higher-order nucleic acid constructs.

High-throughput assembly of genetic elements

The disclosure provides methods and compositions useful for labeling of target molecules with origin-specific nucleic acid identifiers (for example, barcodes), which can be used subsequently to identify, quantify, or otherwise characterize a feature or activity of target molecules originating from a particular discreet volume. Such target molecules can include polypeptides expressed by cells, in which nucleic acid molecules encoding the polypeptides are labeled with the same, or matched, origin-specific nucleic acid identifiers.

Proteomic analysis with nucleic acid identifiers

The invention provides, inter alia, methods for uniquely labeling populations of agents of interest using random combinations of oligonucleotides. The oligonucleotides may comprise a unique nucleotide sequence and/or one or more non-nucleic acid detectable moieties.

Compositions and methods for labeling of agents

The invention is directed to methods for synthesizing oligonucleotides directly on biomolecules or cells living or fixed. In some embodiments, template-free enzymatic synthesis is implemented under biological conditions with successive cycles of (i) enzymatic addition of a 3'-O-blocked nucleoside triphosphate and (ii) enzymatic deblocking of the incorporated nucleotide to regenerate a free 3&#x2032; hydroxyl. The invention has applications in single-cell cDNA library construction and analysis.

Direct oligonucleotide synthesis on cells and biomolecules

The present invention relates to systems and methods for manipulating droplets within a high through put microfluidic system.

High-throughput dynamic reagent delivery system

The present disclosure generally relates to methods and compositions of linking, amplifying, and sequencing nucleic acid molecules. Also disclosed is the use of 5'-5&#x2032;linked oligonucleotides for linking nucleic acid molecules for sequencing of the ends of long nucleic acid template molecules, or for sequencing polymorphism or different target genes or different RNAs simultaneously.

Methods for linking polynucleotides

The present disclosure, in some aspects, provides compositions, systems and methods for proximity-based detection of target biomolecules of interest.

Identification of biomolecular interactions

The present invention is related to genomic nucleotide sequencing. In particular, the invention describes a single reaction method to co-amplify multiple subsequences of a nucleic acid fragment sequence (i.e., for example, at least two read pairs from a single library insert sequence). Nucleic acid fragment sequences may include, but are not limited to, localizing library insert sequences and/or unique read pair sequences in specific orientations on a single emulsion polymerase chain reaction bead. Methods may include, but are not limited to, annealing, melting, digesting, and/or reannealing high throughput sequencing primers to high throughput sequencing primer binding sites. The compositions and methods disclosed herein contemplate sequencing complex genomes, amplified genomic regions, as well as detecting chromosomal structural rearrangements that are compatible with massively parallel high throughput sequencing platforms as well as ion semiconductor matching sequencing platforms (i.e., for example, Ion Torrent platforms).

Compositions and methods for co-amplifying subsequences of a nucleic acid fragment sequence

The present invention is related to genomic nucleotide sequencing. In particular, the invention describes a paired end sequencing method that enables the sequencing of unique read pairs by co-localizing both 5' ends on a single emulsion polymerase chain reaction bead. The method may use a customized forked adaptor primer pair that is compatible with massively parallel sequencing techniques. The compositions and methods disclosed herein contemplate sequencing complex genomes, amplified genomic regions, as well as detecting chromosomal structural rearrangements.

Paired end bead amplification and high throughput sequencing

Growing community of inventors

Cambridge, MA, United States of America

Robert Nicol