Sample indexing for single cells
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.
1. A method for cell identification, comprising:
(a) contacting a first plurality of cells and a second plurality of cells with two sample indexing compositions, respectively,
wherein each of the first plurality of cells and each of the second plurality of cells comprises one or more cellular component targets,
wherein each of the two sample indexing compositions comprises a cellular component binding reagent associated with a sample indexing oligonucleotide,
wherein the cellular component binding reagent is capable of specifically binding to at least one of the one or more cellular component targets, and
wherein the sample indexing oligonucleotide comprises a sample indexing sequence, and wherein sample indexing sequences of the two sample indexing compositions comprise different sequences;
(b) barcoding the sample indexing oligonucleotides using a plurality of barcodes to create a plurality of barcoded sample indexing oligonucleotides,
wherein the plurality of barcodes each comprises a cell label sequence, and wherein at least two of the plurality of barcodes comprise an identical cell label sequence;
(c) obtaining sequencing data of the plurality of barcoded sample indexing oligonucleotides;
(d) identifying a cell label sequence associated with two or more sample indexing sequences in the sequencing data obtained; and
(e) removing sequencing data associated with the cell label sequence identified in step (d) from the sequencing data obtained.
2. The method of claim 1 , wherein sample indexing sequences of at least 10 of the plurality of sample indexing compositions comprise different sequences.
3. The method of claim 1 , wherein the cellular component binding reagent comprises an antibody, a tetramer, an aptamers, a protein scaffold, or a combination thereof.
4. The method of claim 1 , wherein the at least one of the one or more cellular component targets is a cell surface component.
5. The method of claim 1 , wherein the at least one of the one or more cellular component targets comprises a carbohydrate, a lipid, a protein, an extracellular protein, a cell-surface protein, a cell marker, a B-cell receptor, a T-cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an intracellular protein or any combination thereof.
6. The method of claim 1 , further comprising removing unbound sample indexing compositions of the two sample indexing compositions after step (a).
7. The method of claim 1 , wherein the sample indexing oligonucleotide is detachably associated with the cellular component binding reagent.
8. The method of claim 7 , comprising disassociating the sample indexing oligonucleotide from the cellular component binding reagent.
9. The method of claim 1 , wherein the sample indexing oligonucleotide is not homologous to genomic sequences of any of the first plurality of cells and/or the second plurality of cells.
10. The method of claim 1 , wherein the sample indexing composition of the plurality of sample indexing compositions comprises a second cellular component binding reagent not conjugated with the sample indexing oligonucleotide.
11. The method of claim 10 , where the cellular component binding reagent and the second cellular component binding reagent are identical.
12. The method of claim 1 ,
wherein each of the plurality of barcodes comprises one or more of a target-binding region, a molecular label sequence and a binding site for a universal primer, and
wherein molecular label sequences of at least two of the plurality of barcodes comprise different molecule label sequences.
13. The method of claim 12 , wherein the molecular label sequences of the barcodes comprise random sequences.
14. The method of claim 12 , wherein the sample indexing oligonucleotide comprises a sequence complementary to the target-binding region, or a portion thereof, of at least one barcode of the plurality of barcodes.
15. The method of claim 12 , wherein the target-binding region or a portion thereof comprises a poly(dT) sequence.
16. The method of claim 12 , wherein the plurality of barcodes is associated with a particle.
17. The method of claim 16 , wherein the plurality of barcodes is immobilized on the particle, partially immobilized on the particle, enclosed in the particle, partially enclosed in the particle, or a combination thereof.
18. The method of claim 16 , wherein the particle is disruptable.
19. The method of claim 16 , wherein the particle is a sepharose bead, a streptavidin bead, an agarose bead, a magnetic bead, a hydrogel bead a conjugated bead, a protein A conjugated bead, a protein G conjugated bead, a protein A/G conjugated bead, a protein L conjugated bead, an oligo(dT) conjugated bead, a silica bead, a silica-like bead, an anti-biotin microbead, an anti-fluorochrome microbead, or any combination thereof.
20. The method of claim 16 , wherein one or more of the cellular component binding reagent, the particle and the sample indexing oligonucleotide is associated with a detectable moiety.
21. The method of claim 1 , wherein barcoding the sample indexing oligonucleotides using the plurality of barcodes comprises:
contacting the plurality of barcodes with the sample indexing oligonucleotides to generate barcodes hybridized to the sample indexing oligonucleotides; and
extending the barcodes hybridized to the sample indexing oligonucleotides to generate the plurality of barcoded sample indexing oligonucleotides.
22. The method of claim 21 , wherein extending the barcodes comprises extending the barcodes using a DNA polymerase to generate the plurality of barcoded sample indexing oligonucleotides, or extending the barcodes using a reverse transcriptase to generate the plurality of barcoded sample indexing oligonucleotides.
23. The method of claim 21 , comprising amplifying the plurality of barcoded sample indexing oligonucleotides to produce a plurality of amplicons.
24. The method of claim 23 , wherein amplifying the plurality of barcoded sample indexing oligonucleotides comprises amplifying, using polymerase chain reaction (PCR), at least a portion of the molecular label sequence and at least a portion of the sample indexing oligonucleotide.
25. The method of claim 23 , wherein obtaining the sequencing data of the plurality of barcoded sample indexing oligonucleotides comprises obtaining sequencing data of the plurality of amplicons.
26. The method of claim 1 , further comprising identifying the sample origin of the at least one cell comprises identifying sample origin of the plurality of barcoded targets based on the sample indexing sequence of the at least one barcoded sample indexing oligonucleotide.