OLIGONUCLEOTIDES ASSOCIATED WITH ANTIBODIES
Disclosed herein include systems, methods, compositions, and kits for determining protein expression and gene expression simultaneously and for sample indexing. In some embodiments, an oligonucleotide associated with a cellular component-binding reagent (e.g., an antibody) comprises one or more of a unique molecular label sequence, a primer adapter, antibody-specific barcode sequence, an alignment sequence, and/or a poly(A) sequence. In some embodiments, the oligonucleotide is associated with the cellular component-binding reagent via a linker (e.g., 5AmMC12).
1 - 21 . (canceled)
22 . A composition, comprising a first cellular component-binding reagent and a second cellular component-binding reagent,
wherein the first cellular component-binding reagent is capable of specifically binding to a first cellular component target and is associated with a first sample indexing oligonucleotide, wherein the first sample indexing oligonucleotide comprises a first sample indexing sequence and an alignment sequence adjacent to a poly(dA) region,
wherein the second cellular component-binding reagent is capable of specifically binding to a second cellular component target and is associated with a second sample indexing oligonucleotide, wherein the second sample indexing oligonucleotide comprises a second sample indexing sequence and an alignment sequence adjacent to a poly(dA) region, and
wherein the first sample indexing sequence and the second sample indexing sequence are the same, and wherein the first cellular component target and the second cellular component target are different.
23 . The composition of claim 22 , wherein the first sample indexing oligonucleotide and the second sample indexing oligonucleotide comprise one or both of:
(a) a linker, wherein the first sample indexing oligonucleotide and the second sample indexing oligonucleotide are associated with the first cellular component-binding reagent and the second cellular component-binding reagent, respectively, through the linker; and
(b) the sequence of a first universal primer, a complementary sequence thereof, a partial sequence thereof, or a combination thereof.
24 . The composition of claim 23 , wherein the first universal primer is 5-50 nucleotides in length.
25 . The composition of claim 23 , wherein the first universal primer comprises an amplification primer, a sequencing primer, or a combination thereof.
26 . The composition of claim 25 , wherein the sequencing primer comprises a P7 sequencing primer.
27 . The composition of claim 22 , wherein the alignment sequence is one or more nucleotides in length.
28 . The composition of claim 22 , wherein the alignment sequence is two or more nucleotides in length.
29 . The composition of claim 22 , wherein the alignment sequence comprises a guanine, a cytosine, a thymine, a uracil, or a combination thereof.
30 . The composition of claim 22 , wherein the alignment sequence comprises a poly(dT) sequence, a poly(dG) sequence, a poly(dC) sequence, a poly(dU) sequence, or a combination thereof.
31 . The composition of claim 22 , wherein the alignment sequence is 5′ to the poly(dA) region.
32 . The composition of claim 23 , wherein the linker comprises a carbon chain, and wherein the carbon chain comprises 2-30 carbons.
33 . The composition of claim 23 , wherein the linker comprises 5′ amino modifier C12 (5AmMC12), or a derivative thereof.
34 . The composition of claim 22 , wherein the first cellular component target and/or the second cellular component target is, or comprises, an extracellular protein, an intracellular 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 integrin, or any combination thereof.
35 . The composition of claim 22 , wherein the first cellular component target and/or the second cellular component target is ADAM10, CD156c, ANO6, ATP1B2, ATP1B3, BSG, CD147, CD109, CD230, CD29, CD298, ATP1B3, CD44, CD45, CD47, CD51, CD59, CD63, CD97, CD98, SLC3A2, CLDND1, HLA-ABC, ICAM1, ITFG3, MPZL1, NA K ATPase alpha1, ATP1A1, NPTN, PMCA ATPase, ATP2B1, SLC1A5, SLC29A1, SLC2A1, SLC44A2, or any combination thereof.
36 . A method for sample identification, comprising:
contacting each of a plurality of samples with a sample indexing composition of a plurality of sample indexing compositions, respectively,
wherein each of the plurality of samples comprises one or more cells each comprising one or more cellular component targets, wherein the sample indexing composition 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,
wherein the sample indexing oligonucleotide comprises a sample indexing sequence and an alignment sequence adjacent to a poly(dA) region, and wherein sample indexing sequences of at least two sample indexing compositions of the plurality of sample indexing compositions comprise different sequences; and
identifying the sample origin of at least one cell of the one or more cells based on the sample indexing sequence of at least one sample indexing oligonucleotide of the plurality of sample indexing compositions.
37 . The method of claim 36 , wherein identifying the sample origin of the at least one cell comprises:
barcoding sample indexing oligonucleotides of the plurality of sample indexing compositions using a plurality of barcodes to generate a plurality of barcoded sample indexing oligonucleotides;
obtaining sequencing data of the plurality of barcoded sample indexing oligonucleotides; and
identifying the sample origin of the cell based on the sample indexing sequence of at least one barcoded sample indexing oligonucleotide of the plurality of barcoded sample indexing oligonucleotides in the sequencing data, wherein identifying the sample origin of the at least one cell comprises identifying the presence or absence of the sample indexing sequence of at least one sample indexing oligonucleotide, or of at least one barcoded sample indexing oligonucleotide, in the sequencing data.
38 . The method of claim 36 , wherein the sample indexing oligonucleotide is not homologous to genomic sequences of any of the one or more cells, is homologous to genomic sequences of a species, or a combination thereof, and wherein the species is a non-mammalian species.
39 . The method of claim 36 , wherein the sample indexing oligonucleotide comprises one or both of:
(a) a linker, wherein the sample indexing oligonucleotide is associated with the cellular component-binding reagent through the linker; and
(b) the sequence of a first universal primer, a complementary sequence thereof, a partial sequence thereof, or a combination thereof, wherein the first universal primer comprises an amplification primer, a sequencing primer, or a combination thereof.
40 . The method of claim 36 , wherein the alignment sequence is one or more nucleotides in length, and wherein the alignment sequence comprises a guanine, a cytosine, a thymine, a uracil, or a combination thereof.
41 . The method of claim 36 , wherein the alignment sequence is 5′ to the poly(dA) region.