Methods and compositions for identifying or quantifying targets in a biological sample

Abstract

Compositions, kits and methods are described that comprise one or more constructs, each construct comprising a ligand attached or conjugated to a polymer construct, e.g., an oligonucleotide sequence, by a linker, each ligand binding specifically to a single target located in or on the surface of a cell. The polymer construct comprises a) an Amplification Handle; b) a Barcode that specifically identifies a single ligand; c) an optional Unique Molecular Identifier that is positioned adjacent to the Barcode on its 5′ or 3′ end; and d) an Anchor for hybridizing to a complementary sequence, e.g., for generation of a double-stranded oligonucleotide. These compositions are used in methods, including high throughput methods, for detecting one or more targets or epitopes in a biological sample. These compositions are also used in a high throughput method for characterizing a cell by simultaneous detection of one or more epitopes located in or on the cell and its transcriptome.

Description

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0001]This invention was made with government support under Grant Nos. R21-HG-009748 awarded by the National Institutes of Health. The government has certain rights in this invention.INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM[0002]Applicant hereby incorporates by reference the Sequence Listing material filed in electronic form herewith. This file is labeled “NYG_LIPP35US_ST25.txt”, dated January 23, 2018 and contains 11kB.BACKGROUND OF THE INVENTION[0003]The ability to characterize individual cells in a heterogeneous population is becoming increasingly important in biological research and clinical diagnostics. The unbiased and high-throughput nature of modern single cell RNA-seq (scRNA-seq) approaches has proven invaluable for describing heterogeneous cell populations1-3. Prior to single-cell genomics, cellular states were routinely described using curated panels of fluorescently labeled antibodi...

AI Technical Summary

Benefits of technology

This patent describes a method for labeling cells using a special construct that includes a barcode, a random molecular tag, and an anchor sequence. This method allows for the determination of most doublets and control for batch effects in scRNA-seq or CITE-seq methods. The technical effect of this patent is the improved accuracy and reliability of cell analysis using multiplexing in labeling.

Problems solved by technology

However, massively parallel approaches based on droplet microfluidics1-3, microwells47,48 or combinatorial indexing20,30 are incompatible with cytometry and therefore cannot be augmented with protein information.

While effective, FACS / scRNA-seq approaches suffer from relatively low throughput and from an experimental bias in that only cell types chosen a priori are sorted and sequenced.

Thus, these methods are not well suited for discovery of novel cell populations or for characterizing complex tissues that require the analysis of tens of thousands of cells.

Although droplet-based advances in single-cell genomics have dramatically changed the scale of scRNA-seq experiments, these methods suffer from a key disadvantage: All droplet-based single-cell RNA-sequencing methods lose important phenotypic information other than protein levels in general or cell-surface protein expression in particular (Table 1).

These assays are limited in scale and / or can only profile a few genes and proteins in parallel (see Table 1 for comparison of different technologies).

Although the transcriptome can serve as a detailed readout of cellular state, it has been shown that mRNA abundance is often a poor proxy for protein levels especially in developmental processes4-6.

However, FACS-based approaches are limited in terms of the number of markers that can be assayed simultaneously and by the fact that cells chosen for analysis are biased by the selection of known surface markers.

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