Method and apparatus for simultaneous targeted sequencing of dna, rna and protein

Abstract

Provided herein are methods and systems for the simultaneous targeted detection and sequencing of DNA, RNA, and Protein. In typical embodiments, the DNA, RNA, and Proteins are detected, characterized, and sequenced using just a single mammalian cell. One embodiment of detecting and characterizing DNA, RNA, or protein from a mammalian cell includes encapsulating a single cell in a drop and performing a protease digest on the encapsulated cell drop, performing a reverse transcriptase reaction; performing a droplet merger with barcoding PCR reagents and barcoding beads; performing a PCR reaction to attach the cell barcodes to the DNA targeted amplicons, RNA targeted amplicons, and protein tag amplicons, where all amplicons from the same emulsion contain the same cell barcode; and detecting and characterizing a DNA, RNA, or protein amplicon by sequencing the cell barcode incorporated into each amplicon.

1 - 20 . (canceled) 
     
     
         21 . A multiomic detection method for detecting DNA, RNA, and protein from a cell, comprising:
 encapsulating a cell and antibody-oligonucleotide tags in a drop, wherein antibodies of the antibody-oligonucleotide tags exhibit specificity for proteins of the cell, and wherein the drop comprises a reaction mixture comprising a protease;   performing a protease digest on the encapsulated cell drop with the protease to produce a cell lysate, wherein the cell lysate comprises DNA, cDNA, and oligonucleotide tags derived from the antibody-oligonucleotide tags;   combining cell lysate comprising DNA, cDNA, and oligonucleotide tags with barcoding reagents;   performing a nucleic acid amplification reaction using the barcoding reagents to attach cell barcodes to amplicons derived from the DNA, cDNA, and oligonucleotide tags; and   analyzing DNA, RNA, and protein of the cell.   
     
     
         22 . The method of  claim 21 , wherein the DNA and protein of the cell are detected simultaneously. 
     
     
         23 . The method of  claim 21 , further comprising:
 prior to performing the protease digest, providing a reverse transcriptase and generating the cDNA by performing a reverse transcription reaction on the RNA.   
     
     
         24 . The method of  claim 23 , wherein the reverse transcription reaction is performed in the same drop as the protease digest. 
     
     
         25 . The method of  claim 23 , wherein the reverse transcriptase reaction is performed on a PCR thermocycler. 
     
     
         26 . The method of  claim 23 , wherein providing the reverse transcriptase and generating the cDNA further comprises using reverse transcriptase enzyme with RNA-specific primers to extend RNA and generate cDNA templates for detecting the RNA. 
     
     
         27 . The method of  claim 21 , wherein the multiomic detection comprises a triomic interrogation process for the cell. 
     
     
         28 . The method of  claim 21 , wherein the nucleic acid amplification reaction is used to amplify the DNA and antibody-oligonucleotide tags. 
     
     
         29 . The method of  claim 21 , wherein the barcoding reagents are barcoding PCR reagents. 
     
     
         30 . The method of  claim 21 , further comprising performing an exonuclease reaction and a cleanup to separate the amplicons derived from the oligonucleotide tags from the amplicons derived from the DNA. 
     
     
         31 . The method of  claim 30 , wherein performing the exonuclease reaction and the cleanup generates supernatant for processing into protein libraries. 
     
     
         32 . The method of  claim 30 , wherein performing the exonuclease reaction and the cleanup generates beads comprising the amplicons derived from DNA. 
     
     
         33 . The method of  claim 30 , further comprising separating the amplicons derived from DNA and cDNA using a biotin capture oligo and streptavidin beads. 
     
     
         34 . The method of  claim 21 , further comprising making a protein library. 
     
     
         35 . The method of  claim 21 , further comprising making a DNA library. 
     
     
         36 . The method of  claim 21 , further comprising making an RNA library. 
     
     
         37 . The method of  claim 21 , further comprising making DNA and protein libraries. 
     
     
         38 . The method of  claim 37 , further comprising determining an identity of an analyte by purifying and quantifying the DNA and protein libraries. 
     
     
         39 . The method of  claim 21 , wherein the amplicons are selectively purified by a bead purification process. 
     
     
         40 . The method of  claim 21 , wherein the amplicons derived from the DNA, cDNA, and oligonucleotide tags comprise at least one amplicon derived from the DNA, at least one amplicon derived from the cDNA, and at least one amplicon derived from the oligonucleotide tags.