Measurement of protein expression using reagents with barcoded oligonucleotide sequences

Abstract

Some embodiments disclosed herein provide a plurality of compositions each comprising a protein binding reagent conjugated with an oligonucleotide. The oligonucleotide comprises a unique identifier for the protein binding reagent it is conjugated with, and the protein binding reagent is capable of specifically binding to a protein target. Further disclosed are methods and kits for quantitative analysis of a plurality of protein targets in a sample and for simultaneous quantitative analysis of protein and nucleic acid targets in a sample. Also disclosed herein are systems and methods for preparing a labeled biomolecule reagent, including a labeled biomolecule agent comprising a protein binding reagent conjugated with an oligonucleotide.

1 - 404 . (canceled) 
     
     
         405 . A method for sequencing control, comprising:
 contacting one or more cells of a plurality of cells with a control composition of a plurality of control compositions, 
 wherein a cell of the plurality of cells comprises a plurality of targets and a plurality of protein targets, 
 wherein each of the plurality of control compositions comprises a protein binding reagent associated with a control oligonucleotide, wherein the protein binding reagent is capable of specifically binding to at least one of the plurality of protein targets, and wherein the control oligonucleotide comprises a control barcode sequence and a pseudo-target region comprising a sequence substantially complementary to the target-binding region of at least one of the plurality of barcodes; 
 barcoding the control oligonucleotides using a plurality of barcodes to create a plurality of barcoded control oligonucleotides, 
 wherein each of the plurality of barcodes comprises a cell label sequence, a barcode sequence, and a target-binding region, wherein the barcode sequences of at least two barcodes of the plurality of barcodes comprise different sequences, and wherein at least two barcodes of the plurality of barcodes comprise an identical cell label sequence; 
   obtaining sequencing data of the plurality of barcoded control oligonucleotides;   determining at least one characteristic of the one or more cells using at least one characteristic of the plurality of barcoded control oligonucleotides in the sequencing data.   
     
     
         406 . The method of  claim 405 , wherein the pseudo-target region comprises a poly(dA) region. 
     
     
         407 - 417 . (canceled) 
     
     
         418 . The method of  claim 405  wherein the control barcode sequences of at least two of the plurality of control particle oligonucleotides are identical. 
     
     
         419 - 421 . (canceled) 
     
     
         422 . The method of  claim 405  wherein at least two of the plurality of control particle oligonucleotides comprises different control barcode sequences. 
     
     
         423 - 425 . (canceled) 
     
     
         426 . The method of  claim 405  wherein determining the at least one characteristic of the one or more cells comprises: 
 determining the number of cell label sequences with distinct sequences associated with the plurality of barcoded control oligonucleotides in the sequencing data; and 
 determining the number of the one or more cells using the number of cell label sequences with distinct sequences associated with the plurality of barcoded control oligonucleotides. 
 
     
     
         427 . The method of  claim 426 , comprising determining single cell capture efficiency based the number of the one or more cells determined or based on the ratio of the number of the one or more cells determined and the number of the plurality of cells. 
     
     
         428 . (canceled) 
     
     
         429 . The method of  claim 405  wherein determining the at least one characteristic of the one or more cells using the characteristics of the plurality of barcoded control oligonucleotides in the sequencing data comprises: 
 for each cell label in the sequencing data, determining the number of barcode sequences with distinct sequences associated with the cell label and the control barcode sequence; and 
 determining the number of the one or more cells using the number of barcode sequences with distinct sequences associated with the cell label and the control barcode sequence. 
 
     
     
         430 . The method of  claim 429 , wherein determining the number of barcode sequences with distinct sequences associated with the cell label and the control barcode sequence comprises: 
 for each cell label in the sequencing data, determining the number of barcode sequences with the highest number of distinct sequences associated with the cell label and the control barcode sequence.   
     
     
         431 . The method of  claim 430 , wherein determining the number of the one or more cells using the number of barcode sequences with distinct sequences associated with the cell label and the control barcode sequence comprises: 
 generating a plot of the number of barcode sequences with the highest number of distinct sequences with the number of cell labels in the sequencing data associated with the number of barcode sequences with the highest number of distinct sequences; and   determining a cutoff in the plot as the number of the one or more cells.   
     
     
         432 . The method of  claim 405  , wherein the control oligonucleotide is not homologous to genomic sequences of any of the plurality of cells, or wherein the control oligonucleotide is homologous to genomic sequences of a species. 
     
     
         433 . (canceled) 
     
     
         434 . (canceled) 
     
     
         435 . (canceled) 
     
     
         436 . (canceled) 
     
     
         437 . The method of  claim 405  , comprising releasing the control oligonucleotide from the protein binding reagent prior to barcoding the control oligonucleotides. 
     
     
         438 . (canceled) 
     
     
         439 . The method of  claim 405  , wherein at least one of the plurality of binding targets comprises 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. 
     
     
         440 . (canceled) 
     
     
         441 . (canceled) 
     
     
         442 . (canceled) 
     
     
         443 . The method of  claim 405  , wherein the control oligonucleotide is conjugated to the protein binding reagent through a linker. 
     
     
         444 . (canceled) 
     
     
         445 . (canceled) 
     
     
         446 . (canceled) 
     
     
         447 . (canceled) 
     
     
         448 . (canceled) 
     
     
         449 . (canceled) 
     
     
         450 . The method of  claim 405  ,wherein a second protein binding reagent of the plurality of control compositions is not associated with the control oligonucleotide. 
     
     
         451 . The method of  claim 450 , where the protein binding reagent and the second protein binding reagent are identical. 
     
     
         452 . (canceled) 
     
     
         453 . (canceled) 
     
     
         454 . The method of  claim 405  , wherein the plurality of barcodes is associated with a barcoding particle. 
     
     
         455 - 467 . (canceled) 
     
     
         468 . The method of  claim 454  wherein the barcode sequences of the barcodes comprise random sequences. 
     
     
         469 . (canceled) 
     
     
         470 . The method of  claim 405  , wherein barcoding the control oligonucleotides comprises: barcoding the control oligonucleotides using a plurality of barcodes to create a plurality of barcoded control oligonucleotides. 
     
     
         471 . The method of  claim 405  wherein barcoding the plurality of control oligonucleotides using the plurality of barcodes comprises: 
 contacting the plurality of barcodes with control oligonucleotides of the plurality of control compositions to generate barcodes hybridized to the control oligonucleotides; and 
 extending the barcodes hybridized to the control oligonucleotides to generate the plurality of barcoded control oligonucleotides. 
 
     
     
         472 . (canceled) 
     
     
         473 . (canceled) 
     
     
         474 . (canceled) 
     
     
         475 . The method of  claim 473 ,wherein obtaining the sequencing data comprises obtaining sequencing data of the plurality of amplicons and/or sequencing the at least a portion of the barcode sequence and the at least a portion of the control oligonucleotide. 
     
     
         476 - 1021 . (canceled)