Single Cell Genomic Sequencing Using Hydrogel Based Droplets

Abstract

The present disclosure provides ultrahigh-throughput single cell genomic sequencing methods, referred to herein as “SiC-seq”, which methods include encapsulating single cells in molten gel droplets to facilitate bulk cell lysis and purification of genomic DNA in microgels. Systems and devices for practicing the subject methods are also provided.

1 . A method of sequencing single cell genomic DNA, the method comprising:
 encapsulating a population of single cells in molten gel droplets to provide a population of molten gel droplets, wherein each molten gel droplet of the population contains zero or one cell;   solidifying the population of molten gel droplets to provide a population of solidified microgel droplets;   breaking the emulsions of the solidified microgel droplets to provide a population of solidified microgels;   exposing the population of solidified microgels in bulk to lysis conditions sufficient to lyse cells contained within the population of solidified microgels;   purifying genomic DNA from cells contained within the population of solidified microgels in bulk to provide a population of solidified microgels comprising purified genomic DNA;   encapsulating the population of solidified microgels comprising purified genomic DNA into droplets to provide a population of purified genomic DNA-containing droplets;   fragmenting the purified genomic DNA within the population of purified genomic DNA-containing droplets to provide a population of fragmented genomic DNA-containing droplets;   barcoding the fragmented genomic DNA or an amplification product thereof in the population of fragmented genomic DNA-containing droplets to provide a population of barcoded, fragmented genomic DNA-containing droplets;   purifying barcoded, fragmented genomic DNA from the barcoded, fragmented genomic DNA-containing droplets to provide purified, barcoded, fragmented genomic DNA; and   sequencing the purified, barcoded, fragmented genomic DNA.   
     
     
         2 . The method of  claim 1 , wherein the barcoding comprises merging each of the fragmented genomic DNA-containing droplets with a barcode containing droplet. 
     
     
         3 . The method of  claim 2 , wherein each of the barcode containing droplets comprises a unique nucleic acid barcode sequence. 
     
     
         4 . The method of  claim 1 , wherein the method comprises incorporating an adaptor nucleic acid sequence into the fragmented genomic DNA. 
     
     
         5 . The method of  claim 1 , wherein the population of single cells comprises eukaryotic cells. 
     
     
         6 . The method of  claim 5 , wherein the population of single cells comprises mammalian cells. 
     
     
         7 . The method of  claim 1 , wherein the population of single cells comprises bacterial cells. 
     
     
         8 . The method of  claim 1 , wherein the population of single cells comprises fungal cells. 
     
     
         9 . The method of  claim 1 , wherein the molten gel droplet comprises a hydrogel polymer. 
     
     
         10 . The method of  claim 9 , wherein the hydrogel polymer comprises a thermoresponsive polymer. 
     
     
         11 . The method of  claim 10 , wherein the thermoresponsive polymer is agarose. 
     
     
         12 . The method of  claim 1 , wherein the solidifying comprises cooling the population of molten gel droplets. 
     
     
         13 . The method of  claim 1 , wherein the molten gel droplet comprises polyethylene glycol (PEG). 
     
     
         14 . The method of  claim 13 , wherein the solidifying comprises chemically crosslinking the PEG. 
     
     
         15 . The method of  claim 13 , wherein the solidifying comprises photo-crosslinking the PEG. 
     
     
         16 . The method of  claim 1 , wherein the molten gel droplet comprises acrylamide. 
     
     
         17 . The method of  claim 16 , wherein the solidifying comprises chemically crosslinking the acrylamide. 
     
     
         18 . The method of  claim 16 , wherein the solidifying comprises photo-crosslinking the acrylamide. 
     
     
         19 . The method of  claim 1 , wherein the molten gel droplet comprises alginate. 
     
     
         20 . The method of  claim 19 , wherein the solidifying comprises adding calcium to the molten gel droplet. 
     
     
         21 . The method of  claim 1 , wherein the solidified microgels comprise pores sized to retain genomic DNA within the solidified microgels. 
     
     
         22 . The method of  claim 1 , wherein the step of encapsulating the population of single cells in molten gel droplets comprises the addition of an oil. 
     
     
         23 . The method of  claim 1 , wherein the exposing comprises contacting the population of solidified microgels in bulk with a lytic enzyme to lyse cells contained within the population of solidified microgels. 
     
     
         24 . The method of  claim 23 , wherein the lytic enzyme is selected from zymolyase, lysostaphin, mutanolysin, lysozyme, or a combination thereof. 
     
     
         25 . The method of  claim 1 , wherein the step of purifying genomic DNA from cells contained within the population of solidified microgels comprises contacting the population of solidified microgels with a detergent to solubilize cellular material contained within the population of solidified microgels. 
     
     
         26 . The method of  claim 25 , wherein the detergent is selected from lithium dodecyl sulfate, sodium dodecyl sulfate, or a combination thereof. 
     
     
         27 . The method of  claim 1 , wherein the step of purifying genomic DNA from cells contained within the population of solidified microgels comprises contacting the population of solidified microgels with a protease to digest cellular proteins contained within the population of solidified microgels. 
     
     
         28 . The method of  claim 27 , wherein the protease is proteinase K. 
     
     
         29 . The method of  claim 1 , wherein the step of purifying genomic DNA from cells contained within the population of solidified microgels comprises a step of washing the population of solidified microgels, wherein the step of washing the population of solidified microgels comprises contacting the population of solidified microgels with a washing buffer. 
     
     
         30 . The method of  claim 29 , wherein each of the population of purified genomic DNA-containing droplets comprises a complex comprising a transposase and a transposon. 
     
     
         31 . The method of  claim 30 , wherein the step of fragmenting comprises contacting the purified genomic DNA with a complex comprising a transposase and a transposon. 
     
     
         32 . The method of  claim 31 , wherein the complex comprises a transposon that comprises an adapter sequence. 
     
     
         33 . The method of  claim 32 , wherein contacting the purified nucleic acids with the complex provides fragmented genomic DNA comprising the adapter sequence. 
     
     
         34 . The method of  claim 1 , wherein the step of encapsulating a population of single cells in molten gel droplets and the step of barcoding the fragmented genomic DNA or an amplification product thereof are performed using a microfluidic device. 
     
     
         35 . The method of  claim 1 , wherein one or more of the steps of
 solidifying the population of molten gel droplets to provide a population of solidified microgel droplets;   breaking the emulsions of the solidified microgel droplets to provide a population of solidified microgels;   exposing the population of solidified microgels in bulk to lysis conditions sufficient to lyse cells contained within the population of solidified microgels;   purifying genomic DNA from cells contained within the population of solidified microgels in bulk to provide a population of solidified microgels comprising purified genomic DNA; and   fragmenting the purified genomic DNA within the population of solidified microgels comprising purified genomic DNA in bulk to provide a population of solidified microgels comprising fragmented genomic DNA, are not performed using a microfluidic device.   
     
     
         36 . The method of  claim 1 , wherein the population of single cells is a heterogeneous population of single celled microorganisms. 
     
     
         37 . (canceled)