Becton Dickinson and Company Limited

Cellular Research, Inc.

Guardant Health, Inc.

Eleen Shum

Christina Chang

Glenn K Fu

Christina Fan

James Ghadiali

Gretchen Yinbon Lam

Nidhanjali Bansal

Craig Betts

Brent S Gaylord

Julie Wilhelmy

Katherine Lazaruk

Jody Martin

Dennis E Prosen

Olaf Zoellner

Elisabeth Marie Walczak

Margaret Nakamoto

Devon Joseph Jensen

Eric Jensen

Jason G Vidal

Jue Fan

David Rosenfeld

Janice H Lai

Devon Jensen

Jennifer Tsai

Sixing Li

Jurg Rohrer

Lisha Deng

Maria Simbirsky

In accordance with some embodiments herein, compositions and methods for accurate barcoding of nucleic acids are described. The compositions and methods can involve a plurality of unique oligonucleotide species comprising unique molecule barcodes. In some embodiments, the molecule barcodes can have a relatively low G content, and can exhibit reduced bias in amplification and analysis.

Accurate molecular barcoding

Disclosed herein include systems, methods, compositions, and kits for sample identification. A sample indexing composition can comprise, for example, a protein binding reagent associated with a sample indexing oligonucleotide. Different sample indexing compositions can include sample indexing oligonucleotides with different sequences. Sample origin of cells can be identified based on the sequences of the sample indexing oligonucleotides. Sample indexing oligonucleotides can be barcoded using barcoded and lengthened using daisy-chaining primers.

Sample indexing for single cells

Disclosed herein are methods and systems for correcting errors in sample amplification, including the errors occurred in determining the number of targets in samples. In some embodiments, the method comprises: stochastically barcoding a plurality of targets in the samples using oligonucleotides comprising stochastic barcodes to generate stochastically barcoded targets; contacting one or more defined barcoded primers with each of the one or more samples; and determining an amplification noise.

Error correction in amplification of samples

Disclosed herein include systems, methods, compositions, and kits for the labeling of nucleic acids targets. In some embodiments, the methods comprise the use of target-specific primers and target-non-specific primers during library preparation (e.g., hybrid library preparation). In some embodiments, the methods increase the abundance of select low abundance targets during cDNA library preparation and/or sequencing library preparation.

Hybrid targeted and whole transcriptome amplification

Disclosed herein are methods, compositions, and kits for barcoding nucleic acids. For example, particles for barcoding are provided, where the particle comprises: a first plurality of oligonucleotide barcodes each comprising a target-binding region, and a second plurality of oligonucleotides each comprising a target-specific target-binding region.

Particles associated with oligonucleotides

Disclosed herein include systems, methods, compositions, and kits for whole transcriptome analysis (WTA) with random priming and extension (RPE). The RPE-based WTA method can comprise hybridizing random primers with a plurality of first strand barcoded polynucleotides associated with a solid support and extending the random primers to generate a plurality of extension products. The method can comprise amplifying the plurality of extension products to generate a sequencing library.

Whole transcriptome analysis of single cells using random priming

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

Disclosed herein include systems, methods, compositions, and kits for quantitative analysis of a plurality of cellular component targets of cells of interest. In some embodiments, an oligonucleotide associated with a cellular component-binding reagent (e.g., an antibody) is associated with one or more detectable moieties (e.g., luminescent moieties, fluorescent moieties, a phosphorescent moieties). In some embodiments, the presence of the detectable moiety in the binding reagent oligonucleotide enables the cellular component-binding reagent to be employed for both fluorescence analysis (e.g., cell sorting) and sequence analysis (e.g., protein expression profiling).

Oligonucleotide-comprising cellular component-binding reagents and methods of using the same

Disclosed herein include systems, methods, compositions, and kits for determining protein expression and gene expression simultaneously and for sample indexing. In some embodiments, an oligonucleotide associated with a cellular component-binding reagent (e.g., an antibody) comprises one or more of a unique molecular label sequence, a primer adapter, antibody-specific barcode sequence, an alignment sequence, and/or a poly(A) sequence. In some embodiments, the oligonucleotide is associated with the cellular component-binding reagent via a linker (e.g., 5AmMC12).

Oligonucleotides associated with antibodies

Disclosed herein include systems, methods, compositions, and kits for labeling nucleic acid targets in a sample. In some embodiments, nucleic acid targets (e.g., mRNAs) are initially barcoded on the 3' end and are subsequently barcoded on the 5&#x2032; end following a template switching reaction and intermolecular and/or intramolecular hybridization and extension. There are provided, in some embodiments, methods of 5&#x2032;-based and 3&#x2032;-based gene expression profiling. Immune repertoire profiling methods are also provided in some embodiments.

Determining 5' transcript sequences

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 extension in single cell whole transcriptome analysis

Some embodiments disclosed herein provide a plurality of compositions each comprising a protein binding reagent conjugated with an oligonucleotide. The oligonucleotide comprises a unique identifier for the protein binding reagent it is conjugated with, and the protein binding reagent is capable of specifically binding to a protein target. Further disclosed are methods and kits for quantitative analysis of a plurality of protein targets in a sample and for simultaneous quantitative analysis of protein and nucleic acid targets in a sample. Also disclosed herein are systems and methods for preparing a labeled biomolecule reagent, including a labeled biomolecule agent comprising a protein binding reagent conjugated with an oligonucleotide.

Measurement of protein expression using reagents with barcoded oligonucleotide sequences

Methods of repairing a partially double-stranded DNA fragment are provided. In some embodiments, the methods comprise (a) contacting the partially double-stranded DNA fragment with one or more primers of a primer population, wherein the partially double-stranded DNA fragment comprises a 3' overhang and the primer population comprises a random target-hybridizing sequence; (b) extending one or more primers of the primer population along the DNA fragment using a DNA polymerase, thereby producing one or more extended primers annealed to the DNA fragment; and (c) ligating the 3&#x2032; end of one or more extended primers to the 5&#x2032; end of an extended primer or a strand of the partially double-stranded DNA fragment, thereby providing a repaired DNA fragment.

Growing community of inventors

Menlo Park, CA, United States of America

Eleen Shum