Sample indexing for single cells
Inventors
Chang, Christina • Fan, Christina • Shum, Eleen • Martin, Jody • Bansal, Nidhanjali • Ghadiali, James • Lazaruk, Katherine • Lam, Gretchen Yinbon
Assignees
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Publication Number
US-10676779-B2
Publication Date
2020-06-09
Expiration Date
Abstract
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.
Core Innovation
The invention provides a multiplexed single-cell workflow in which a first sample and a second sample, each comprising a plurality of cells, are labeled with different sample indexing compositions. Each sample indexing composition comprises a cellular component binding reagent associated with a sample indexing oligonucleotide that specifically binds to a cellular component target of the cells, and the first and second sample indexing oligonucleotides comprise different sample indexing sequences while the cellular component targets are the same.
After labeling, the first labeled sample and the second labeled sample are pooled to form a combined labeled sample containing cells associated with the first sample indexing composition and cells associated with the second sample indexing composition. The pooled cells are partitioned into a plurality of partitions so that each partition comprises a single cell of the combined labeled sample, and the sample indexing oligonucleotide is subjected to one or more reactions to generate a barcoded sample indexing oligonucleotide comprising the corresponding sample indexing sequence or a complement thereof for sequencing.
Sequencing information is obtained for the barcoded sample indexing oligonucleotide or a portion thereof, and cells in the combined labeled sample are determined to be from the first sample or the second sample based on the first sample indexing sequence or the second sample indexing sequence, a complementary sequence thereof, or a portion thereof, in the obtained sequence information. The disclosure also describes stochastic barcoding, daisy-chained or barcode-lengthening amplification, sequencing control concepts, multiplet and cell-counting concepts, and interaction determination compositions that use paired interaction oligonucleotides ligated by a bridge and barcoded to detect protein-protein interactions based on ligation-association sequencing.
Claims Coverage
Independent claim clm-00001 covers a multiplexed single-cell workflow with 6 inventive features: labeling two samples with different sample indexing sequences, pooling and partitioning into single-cell partitions, barcoding the sample indexing oligonucleotides, sequencing, and determining sample origin from sequencing information. No other independent claims are provided in the supplied claim set.
Two-sample cellular-component indexing with different sample indexing sequences
Contacting a first sample and a second sample with first and second sample indexing compositions, wherein each sample indexing composition comprises a cellular component binding reagent associated with a respective sample indexing oligonucleotide having different sample indexing sequences, and wherein the cellular component targets for both pluralities of cells are the same.
Pooling labeled samples into a combined labeled sample
Pooling the first labeled sample and the second labeled sample to form a combined labeled sample comprising cells associated with the first sample indexing composition and cells associated with the second sample indexing composition.
Partitioning combined labeled sample into single-cell partitions
Partitioning the cells of the combined labeled sample into a plurality of partitions, wherein each partition comprises a single cell of the combined labeled sample.
Barcoding sample indexing oligonucleotides to generate barcoded sample indexing oligonucleotides
Subjecting the first sample indexing oligonucleotide or the second sample indexing oligonucleotide to one or more reactions to generate a first barcoded sample indexing oligonucleotide comprising the first sample indexing sequence, or a complement thereof, or a second barcoded sample indexing oligonucleotide comprising the second sample indexing sequence, or a complement thereof.
Sequencing barcoded sample indexing oligonucleotides to obtain sequencing information
Obtaining sequencing information of the first barcoded sample indexing oligonucleotide or the second barcoded sample indexing oligonucleotide, or a portion thereof.
Determining sample origin based on sequencing information of indexing sequences
Determining cells in the combined labeled sample to be from the first sample or the second sample based on the first sample indexing sequence or the second sample indexing sequence, respectively, a complementary sequence thereof, or a portion thereof, in the sequence information obtained.
Claim coverage centers on labeling two samples with cellular component binding reagents associated with different sample indexing sequences, pooling and partitioning into single-cell partitions, generating barcoded sample indexing oligonucleotides via one or more reactions, sequencing the barcoded indexing oligonucleotides, and determining whether each cell originates from the first or second sample based on indexing-sequence readouts.
Stated Advantages
Enhanced throughput
Cost reduction
Batch-effect minimization
Identify cells in the combined labeled sample as originating from the first sample or the second sample based on sequencing information.
Enable multiplexed sample indexing by using different sample indexing sequences associated with different sample indexing compositions.
Support sequencing workflows by generating barcoded sample indexing oligonucleotides and obtaining sequencing information.
Documented Applications
Identifying multiplets during analysis
Multiplex sample indexing to identify cell/sample origin for single cells.
Quantitative analysis and simultaneous protein and nucleic-acid profiling.
Use of control particles or control particle oligonucleotides for sequencing efficiency or normalization.
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