Methods of associating phenotypical data and single cell sequencing data

Patent metadata

Field	Value
Publication number	US-11965208-B2
Application number	US-202016849191-A
Country	US
Kind code	B2
Filing date	Apr 15, 2020
Priority date	Apr 19, 2019
Publication date	Apr 23, 2024
Grant date	Apr 23, 2024

Abstract

Disclosed herein include systems, methods, compositions, and kits for associating single cell sequencing data with phenotypic data and/or agent exposure. In some embodiments, cell indexing oligonucleotides comprising cell indexing sequences are associated with cells. In some embodiments, synthetic particles are associated with stochastic labels comprising cell indexing sequences. Sequential hybridizing and imaging of probes annealing to a cell indexing sequence can generate a spatial index. The spatial index can be employed to identify of the partition from which a sequenced nucleic acid target molecule originated.

First claim

What is claimed is:

1. A method of labeling a plurality of cells with cell indexing oligonucleotides, comprising:
(a) associating cells in each first partition of a plurality of first partitions with a cellular component-binding reagent of a plurality of cellular component-binding reagents,
wherein each of the cells comprises one or more cellular component targets, wherein the cellular component-binding reagent is associated with a first indexing oligonucleotide with a first cell indexing subsequence, wherein cellular component-binding reagents in each first partition of the plurality of partitions are associated with first indexing oligonucleotides with the same first cell indexing subsequences, and the first cell indexing subsequences are different between at least two first partitions, and wherein the cellular component-binding reagent is capable of specifically binding to the one or more cellular component targets,
thereby generating cells associated with the first cell indexing subsequence, in each first partition of the plurality of first partitions, via the cellular component-binding reagent associated with the first indexing oligonucleotide with the first cell indexing subsequence, wherein the cells in at least two first partitions of the plurality of first partitions are associated with first cell indexing subsequences that are different between the at least two first partitions;
(b) pooling the cells associated with the cellular component-binding reagents in the plurality of first partitions;
(c) distributing the pooled cells into a plurality of second partitions;
(d) associating the distributed cells in each second partition of the plurality of second partitions with a second indexing oligonucleotide of a plurality of second indexing oligonucleotides with a second cell indexing subsequence, wherein the second indexing oligonucleotides of the plurality of second indexing oligonucleotides in at least two second partitions of the plurality of second partitions comprise second cell indexing subsequences that are different between the at least two second partitions,
thereby generating cells associated with the second cell indexing subsequence, in each second partition of the plurality of second partitions, wherein the cells in two second partitions of the plurality of second partitions are associated with different second cell indexing subsequences;
(e) generating cells associated with the cellular component-binding reagent in each second partition of the plurality of second partitions associated with a cell indexing oligonucleotide,
wherein a cell indexing sequence of the cell indexing oligonucleotide comprises:
the first cell indexing subsequence; and
the second cell indexing subsequence of the second cell indexing oligonucleotide associated with the second partition of the plurality of second partitions, via the cellular component-binding reagent associated with the first indexing oligonucleotide with the first cell indexing subsequence associated with each of the cells in the second partition;

(f) pooling the cells associated with the cellular component-binding reagents in the plurality of second partitions;
(g) performing steps (c), (d), (e), and (f) n times, wherein n is a positive integer greater than 1,
thereby generating a plurality of cells associated with unique cell indexing oligonucleotides, each unique cell indexing oligonucleotide comprising:
n cell indexing subsequences; and
a target sequence comprising a sequence complementary to a capture sequence of a stochastic label configured to hybridize to the target sequence, wherein the stochastic label comprises a target binding region comprising the capture sequence, wherein the target binding region is configured to hybridize to a nucleic acid target of a cell of the plurality of cells,

wherein different cells of the plurality of cells are associated with cell indexing oligonucleotides comprising different n cell indexing subsequences;
(h) partitioning the plurality of cells associated with the unique cell indexing oligonucleotides to a plurality of third partitions on a substrate, wherein each third partition of the plurality of third partitions is a distinct spatial location on the substrate and comprises at most a single cell from the plurality of cells associated with the unique cell indexing oligonucleotides;
(i) determining a spatial index of the unique cell indexing oligonucleotides in the plurality of third partitions using n pluralities of probes by sequentially hybridizing and imaging the n pluralities of probes annealing to the n cell indexing subsequences, wherein the spatial index identifies a distinct spatial location of each of the plurality of third partitions on the substrate.

2. The method of claim 1 , wherein the nth indexing oligonucleotide comprises an (n−1)th linker sequence and the target sequence.

3. The method of claim 2 , wherein the target sequence comprises a poly(A) sequence.

4. The method of claim 1 , wherein the n pluralities of probes comprise one or more detectable moieties, wherein the imaging comprises detecting the one or more detectable moieties.

5. A method of generating a spatial index, comprising the steps of:
partitioning a plurality of synthetic particles comprising stochastic labels that comprise unique cell indexing oligonucleotides to a plurality of partitions in a substrate, wherein each partition of the plurality of partitions is a distinct spatial location on the substrate and comprises a unique cell indexing oligonucleotide,
wherein the unique cell indexing oligonucleotide comprises a cell indexing sequence comprising a series of n cell indexing subsequences that represent n distinct positions along the cell indexing sequence, wherein n is an integer greater than 1,
wherein each of the plurality of stochastic labels comprises:
a cellular label comprising the cell indexing sequence;
a molecular label, wherein molecular label sequences of at least two stochastic labels of the plurality of stochastic labels comprise different molecule label sequences; and
a target nucleic acid binding region; and

in each partition of the plurality of partitions:
(a) contacting the unique cell indexing oligonucleotide with a plurality of probes,
wherein one or more of the probes of the plurality of probes comprise a detectable moiety, or precursor thereof, wherein at least one of the plurality of probes selectively anneal to cell indexing subsequences with complementary sequences, wherein the at least one probe that selectively anneals to a cell indexing subsequence anneals to at most one of the n distinct positions along the cell indexing sequence;
(b) removing unannealed probes;
(c) imaging the unique cell indexing oligonucleotide, wherein imaging comprises detecting a signal of the detectable moiety at the distinct spatial location on the substrate;
(d) removing the annealed probes and/or the detectable moieties thereof; and
(e) repeating the steps (a)-(d) for a total of n cycles, wherein in each cycle, each probe that selectively anneals to a cell indexing subsequence anneals to a different position of the n distinct positions along the cell indexing sequence than the position of the cell indexing subsequence along the cell indexing sequence to which the at least one probe selectively annealed in any preceding cycle, whereby a spatial index is generated for the unique cell indexing oligonucleotide,

wherein the spatial index comprises a series of: one or more signals; and/or one or more absences of a signal, detected in the n cycles, wherein the sum of the number of the one or more signals and the number of the one or more absences of a signal is n, wherein the spatial index identifies the distinct spatial location on the substrate of each of the plurality of partitions.

Assignees

• Becton Dickinson Co

Inventors

• Chang Christina
• Li Sixing

Classifications

• C12Q1/6837 Primary

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title

• C12Q1/6827

  for detection of mutation or polymorphism · CPC title

• C12Q1/6858

  Allele-specific amplification · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title