Dna sequencing using hydrogel beads

Abstract

Systems, methods, and compositions provided herein relate to preparation of beads encapsulating long DNA fragments for high-throughput spatial indexing. Some embodiments include preparation of nucleic acid libraries within the bead, wherein the bead includes pores that allow diffusion of reagents while retaining genetic material.

1 . (canceled) 
     
     
         2 . A solid support comprising a hydrogel, wherein the hydrogel comprises genetic material, wherein the hydrogel comprises pores that allow diffusion of a reagent through the hydrogel, while retaining the genetic material. 
     
     
         3 . The solid support of  claim 2 , wherein the solid support comprises wells, grooves, or patterns. 
     
     
         4 . The solid support of  claim 3 , wherein the wells are from about 10 μm to about 50 μm in diameter, and wherein the wells are about 0.5 μm to about 1 μm in depth. 
     
     
         5 . The solid support  claim 4 , wherein the wells comprise hydrophobic material comprising an amorphous fluoropolymer. 
     
     
         6 . The solid support of  claim 5 , wherein the amorphous fluoropolymer comprises CYTOP, Fluoropel, or Teflon. 
     
     
         7 . The solid support of  claim 2 , wherein the genetic material comprises single cell microbes. 
     
     
         8 . The solid support of  claim 7 , wherein the microbes are  L. gasseri, S. aureus, B. cereus, B. vulgatus, A. baumannii, S. agalactiae , or  P. acnes.    
     
     
         9 . The solid support of  claim 2 , wherein the reagent comprises enzymes, chemicals, and primers having a size of less than 50 base pairs. 
     
     
         10 . The solid support of  claim 2 , wherein the reagent comprises lysozyme, proteinase K, random hexamers, polymerase, transposase, primers, ligase, catalyzing enzyme, deoxynucleotide triphosphates, buffers, or divalent cations. 
     
     
         11 . The solid support of  claim 2 , wherein the solid support is functionalized with a surface polymer comprising poly(N-(5-azidoacetamidylpentyl) acrylamide-co-acrylamide) (PAZAM) or silane free acrylamide (SFA). 
     
     
         12 . The solid support of  claim 2 , wherein the hydrogel is a hydrogel bead or a hydrogel layer. 
     
     
         13 . The solid support of  claim 2 , wherein the hydrogel comprises polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N,N′-bis(acryloyl)cystamine (BACy), PEG, polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamic acid), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethyleneglycol diallyl ether, ethyleneglycol diacrylate, polymethyleneglycol diacrylate, polyethyleneglycol diacrylate, trimethylopropoane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetracrylate, or combinations or mixtures thereof. 
     
     
         14 . The solid support of  claim 13 , wherein the hydrogel comprises PEG-thiol/PEG-acrylate, acrylamide/N,N′-bis(acryloyl)cystamine (BACy), or PEG/PPO. 
     
     
         15 . A method of identifying single cells, comprising
 providing a solid support comprising hydrogel that comprises genetic material of  claim 2 , wherein the genetic material comprises single cells;   lysing the single cells;   amplifying genetic material from the single cells within the hydrogel;   performing a tagmentation reaction on the genetic material; and   sequencing the genetic material, thereby generating a library encapsulated within the hydrogel.   
     
     
         16 . The method of  claim 15 , further comprising performing an amplification reaction on the genetic material encapsulated within the hydrogel prior to performing the tagmentation reaction. 
     
     
         17 . The method of  claim 16 , wherein the amplification reaction comprises multiple displacement amplification (MDA). 
     
     
         18 . The method of  claim 15 , wherein the tagmentation reaction comprises contacting genetic material with a transposase mixture comprising adapter sequences and transposomes. 
     
     
         19 . The method of  claim 15 , further comprising degrading the hydrogel and depositing the library on the solid support. 
     
     
         20 . The method of  claim 19 , wherein the hydrogel is degraded by contacting the hydrogel with a cleavage mix or by heating the hydrogel to about 90° C. to release the library. 
     
     
         21 . The method of  claim 20 , wherein the cleavage mix comprises dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or tris(3-hydroxypropyl)phosphine (THP).