DIGITAL PROTEIN QUANTIFICATION

• US 20170192013A1
• Filed: 12/29/2016
• Published: 07/06/2017
• Est. Priority Date: 12/30/2015
• Status: Active Grant

• Current Assignee: Bio-Rad Europe GmbH (Bio-Rad Laboratories Incorporated)
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Agresti, Jeremy
• Granted Patent: US 11,965,891 B2
• Time in Patent Office: Days
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: C40B 20/04 Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
• CPC Class Codes: C40B 70/00 Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
• CPC Class Codes: G01N 33/6878 in eptitope analysis

Abstract

Methods and compositions are described for single cell resolution, quantitative proteomic analysis using high throughput sequencing.

Claims

1. A plurality of mixture partitions, wherein the individual mixture partitions comprise:

i) a plurality of fixed proteins, wherein all of the fixed proteins in the individual mixture partition are from one cell; and

ii) a library of at least about 10 structurally distinct antibodies, wherein the structurally distinct antibodies have a specific binding affinity for, and are bound to, structurally distinct target epitopes of the fixed proteins, and wherein the structurally distinct antibodies are conjugated to target-epitope specific oligonucleotides with an optionally cleavable linker, wherein the target-epitope specific oligonucleotides comprise;

a) target-epitope specific barcode sequences, wherein the target-epitope specific barcode sequences are the same for any one structurally distinct antibody and different for all other structurally distinct antibodies; and

b) optionally, unique molecular identifier sequences, wherein the optional unique molecular identifier sequences are different for every molecule of target-epitope specific oligonucleotide.

3. The plurality of mixture partitions of claim 1, wherein the individual mixture partitions further comprise:

iii) a plurality of partition-specific oligonucleotides, the individual partition-specific oligonucleotides comprising a partition-specific barcode sequence that is identical among all partition-specific oligonucleotides of any one mixture partition and different from all partition-specific barcode sequences in other mixture partitions of the plurality, and optionally a unique molecular identifier sequence, wherein the unique molecular identifier sequence is different for every molecule of partition-specific barcode oligonucleotide.

4. The plurality of mixture partitions of claim 3, wherein the partition-specific oligonucleotides of the individual mixture partitions are covalently linked to a bead.

5. The plurality of mixture partitions of claim 3, wherein the partition-specific oligonucleotides of the individual mixture partitions are covalently linked to a bead with a cleavable linker.

6. The plurality of mixture partitions of claim 5, wherein the cleavable linker is selected from the group consisting of a linker comprising a uracil nucleotide, a linker comprising a disulfide linkage, and a linker comprising a restriction endonuclease cleavage site.

7. The plurality of mixture partitions of claim 4, wherein the beads comprise cross-linked agarose.

8. The plurality of mixture partitions of claim 1, wherein the target-epitope specific oligonucleotides are covalently linked to the structurally distinct antibodies with a cleavable linker.

9. The plurality of mixture partitions of claim 8, wherein the cleavable linker is selected from the group consisting of a linker comprising a uracil nucleotide, a linker comprising a disulfide linkage, and a linker comprising a restriction endonuclease cleavage site.

10. The plurality of mixture partitions of claim 1, wherein the individual mixture partitions further comprise a universal primer, wherein the universal primer comprises a 3′

priming region having a universal priming sequence that hybridizes to a universal primer binding site of the target-epitope specific oligonucleotides, or reverse complements thereof, and optionally a unique molecular identifier sequence, wherein the unique molecular identifier sequence is different for every molecule of universal primer.

11. The plurality of mixture partitions of claim 10, wherein the individual mixture partitions comprise the partition-specific oligonucleotides, and wherein the partition-specific oligonucleotides further comprise a 3′

priming region that hybridizes to a partition-specific oligonucleotide primer binding site of the target-epitope specific oligonucleotides, or reverse complements thereof, wherein the 3′

universal primer binding site and the partition-specific oligonucleotide primer binding site of the target-epitope specific oligonucleotides are on opposite strands of a double stranded target-epitope specific oligonucleotide and flank the target-epitope specific barcode sequence, and optionally the unique molecular identifier sequence.

12. The plurality of mixture partitions of claim 11, wherein the 3′

priming region of the partition-specific oligonucleotides is at least 12 and no more than 40 nucleotides in length.

13. The plurality of mixture partitions of claim 10, wherein the universal priming sequence is at least 12 and no more than 25 nucleotides in length.

14. The plurality of mixture partitions of claim 1, wherein the target-epitope specific barcode sequences are at least 4 nucleotides and no more than 100 nucleotides in length.

15. The plurality of mixture partitions of claim 1, wherein the target-epitope specific oligonucleotides comprise unique molecular identifier sequences.

16. The plurality of mixture partitions of claim 15, wherein the unique molecular identifier sequences are at least 4 nucleotides and no more than 15 nucleotides in length.

24. A method for generating the plurality of mixture partitions of claim 3, the method comprising:

i) providing a fixed and permeabilized plurality of single cells, wherein individual fixed and permeabilized single cells comprise the fixed proteins of one single cell;

ii) incubating the fixed and permeabilized plurality of single cells with the library of at least about 10 structurally distinct antibodies conjugated to the target-epitope specific oligonucleotides, thereby binding the antibodies to their corresponding epitopes, if present, to form a plurality of antibody library-single-cell epitope complexes;

iii) washing away unbound antibodies;

iv) partitioning the plurality of antibody library-single-cell complexes into the plurality of mixture partitions, and optionally discarding mixture partitions that do not contain a single cell and/or contain multiple cells; and

v) partitioning a plurality of partition-specific barcode oligonucleotides into the plurality of partitions, and optionally discarding mixture partitions that do not contain a single partition-specific barcode sequence and/or contain multiple partition-specific barcode sequences.

25. The method of claim 24, wherein the method comprises incubating the fixed and permeabilized plurality of single cells with the library of at least about 10, and no more than about 10,000, structurally distinct antibodies conjugated to the target-epitope specific oligonucleotides.

26. The method of claim 24, wherein the method comprises performing iv) before v), and the partition-specific barcode oligonucleotides are partitioned into a plurality of mixture partitions comprising the antibody library-single-cell complexes.

27. The method of claim 24, wherein the method comprises performing v) before iv), and the antibody library-single-cell complexes are partitioned into a plurality of mixture partitions comprising the partition-specific barcode oligonucleotides.

28. The method of claim 24, wherein the partition-specific barcode oligonucleotides are covalently linked to a bead with an optionally cleavable linker.

29. A method for performing single-cell resolution target epitope analysis by high throughput sequencing, the method comprising:

i) forming or providing a plurality of mixture partitions according to claim 11, wherein the mixture partitions further comprise a thermostable polymerase, and wherein;

a) the target-epitope specific oligonucleotides are covalently conjugated to the structurally distinct antibodies with a cleavable linker;

b) the partition-specific oligonucleotides are covalently conjugated to the beads with a cleavable linker;

or c) a) and b); and

ii) cleaving the cleavable linkers of a), b), or c);

iii) firstly hybridizing;

a) the 3′

priming regions of the partition-specific oligonucleotides to 5′

ends of the target-epitope specific oligonucleotides, and extending the hybridized partition specific oligonucleotides with the polymerase, thereby generating double stranded target-epitope specific oligonucleotides comprising the target-epitope specific barcode sequences, the partition-specific barcode sequences, and optionally universal molecular identifier sequences;

or

b) the 3′

priming regions of the universal primers to 5′

ends of the target-epitope specific oligonucleotides, and extending the hybridized universal primers with the polymerase, thereby generating double stranded target-epitope specific oligonucleotides comprising a universal priming region, the target-epitope specific barcode sequences, and optionally universal molecular identifier sequences; and

iv) secondly hybridizing;

a) the 3′

priming regions of the partition-specific oligonucleotides to 5′

ends of the double-stranded target-epitope specific oligonucleotides comprising the universal priming regions, if present, and extending the hybridized partition specific oligonucleotides with the polymerase;

or

b) the 3′

priming regions of the universal primers to 5′

ends of the double-stranded target-epitope specific oligonucleotides comprising the partition-specific barcode sequences, if present, and extending the hybridized universal primers with the polymerase,

thereby generating double stranded target-epitope specific oligonucleotides comprising the universal priming region, the target-epitope specific barcode sequences, the partition-specific barcode sequences, and optionally universal molecular identifier sequences; and

v) amplifying the double stranded target-epitope specific oligonucleotides of iv); and

vi) combining and sequencing the amplified double stranded target-epitope specific oligonucleotides in a high throughput sequencing reaction to obtain a number of target-epitope specific oligonucleotide sequence reads, wherein the sequencing comprises;

a) determining the partition-specific barcode sequence, thereby determining the single cell to which the sequencing data corresponds; and

b) determining the target-epitope specific barcode sequence, thereby determining the protein epitope to which the sequencing data corresponds, wherein the number of target-epitope specific oligonucleotide sequence reads, in which the reads have the same partition-specific barcode sequence and target-epitope specific barcode sequence is proportional to a level of the epitope in the single cell to which the sequencing data corresponds.

30. The method of claim 29, wherein the double stranded target-epitope specific oligonucleotides further comprise the universal molecular identifier sequences, and the method further comprises determining the universal molecular identifier sequence;

and, normalizing the number of target-epitope specific oligonucleotide sequence reads for amplification bias by identifying stranded target-epitope specific oligonucleotide sequences having the same universal molecular identifier sequence as amplification duplicates.

31. The method of claim 29, wherein the double stranded target-epitope specific oligonucleotides further comprise a sample index sequence, and the method further comprises determining the sample index sequence;

and, identifying the source of the single cell to which the sequencing data corresponds.

2. The plurality of mixture partitions, wherein the library contains at least about 10 and no more than about 10,000 structurally distinct antibodies.

17. A plurality of mixture partitions, wherein the individual mixture partitions comprise:

i) a plurality of fixed proteins, wherein all of the fixed proteins in the individual mixture partition are from one cell;

ii) a library of at least about 10 structurally distinct antibodies, wherein the structurally distinct antibodies have a specific binding affinity for, and are bound to, structurally distinct target epitopes of the fixed proteins;

iii) a plurality of double-stranded target-epitope specific oligonucleotides, wherein the individual double-stranded target-epitope specific oligonucleotides are either covalently linked to a corresponding individual structurally distinct antibody, cleaved from the corresponding individual structurally distinct antibody, or comprise a reverse complement of an oligonucleotide covalently linked to or cleaved from the corresponding individual structurally distinct antibody, and wherein the target specific oligonucleotides comprise;

a) target-epitope specific barcode sequences, wherein the target-epitope specific barcode sequences are the same for any one structurally distinct antibody and different for all other structurally distinct antibodies;

b) optional unique molecular identifier sequences, wherein the unique molecular identifier sequences are different for every target-epitope specific oligonucleotide;

c) a partition-specific barcode sequence that is identical among all partition-specific oligonucleotides of any one mixture partition and different from all partition-specific barcode sequences in other mixture partitions of the plurality of mixture partitions;

d) a first 5′

region comprising a first sequencing primer binding region; and

e) a second 5′

region comprising a second sequencing primer binding region, wherein the first and second primer binding regions are on opposite strands of the double stranded target-epitope specific oligonucleotide, are structurally different from each other, and flank the target-epitope specific barcode sequence, partition-specific barcode sequence, and optional universal molecular identifier sequence.

18-23. -23. (canceled)