title: A simple method for encapsulating single cells in alginate microspheres allows for direct PCR and whole genome amplification.
abstract: Microdroplets are an effective platform for segregating individual cells and amplifying DNA. However, a key challenge is to recover the contents of individual droplets for downstream analysis. This paper offers a method for embedding cells in alginate microspheres and performing multiple serial operations on the isolated cells. Rhodobacter sphaeroides cells were diluted in alginate polymer and sprayed into microdroplets using a fingertip aerosol sprayer. The encapsulated cells were lysed and subjected either to conventional PCR, or whole genome amplification using either multiple displacement amplification (MDA) or a two-step PCR protocol. Microscopic examination after PCR showed that the lumen of the occupied microspheres contained fluorescently stained DNA product, but multiple displacement amplification with phi29 produced only a small number of polymerase colonies. The 2-step WGA protocol was successful in generating fluorescent material, and quantitative PCR from DNA extracted from aliquots of microspheres suggested that the copy number inside the microspheres was amplified up to 3 orders of magnitude. Microspheres containing fluorescent material were sorted by a dilution series and screened with a fluorescent plate reader to identify single microspheres. The DNA was extracted from individual isolates, re-amplified with full-length sequencing adapters, and then a single isolate was sequenced using the Illumina MiSeq platform. After filtering the reads, the only sequences that collectively matched a genome in the NCBI nucleotide database belonged to R. sphaeroides. This demonstrated that sequencing-ready DNA could be generated from the contents of a single microsphere without culturing. However, the 2-step WGA strategy showed limitations in terms of low genome coverage and an uneven frequency distribution of reads across the genome. This paper offers a simple method for embedding cells in alginate microspheres and performing PCR on isolated cells in common bulk reactions, although further work must be done to improve the amplification coverage of single genomes.
authors.affiliation: Stanford Genome Technology Center, Department of Biochemistry, Stanford University, 3165 Porter Drive, Palo Alto, CA 94304, United States of America; Department of Chemical Engineering, Stanford University, Stanford, CA, United States of America.
authors.forename: Saharnaz
authors.initials: S
authors.lastname: Bigdeli
authors.affiliation: Stanford Genome Technology Center, Department of Biochemistry, Stanford University, 3165 Porter Drive, Palo Alto, CA 94304, United States of America; Caerus Molecular Diagnostics, Mountain View, CA, United States of America.
authors.forename: Roger O
authors.initials: RO
authors.lastname: Dettloff
authors.affiliation: Department of Chemical Engineering, Stanford University, Stanford, CA, United States of America.
authors.forename: Curtis W
authors.initials: CW
authors.lastname: Frank
authors.affiliation: Stanford Genome Technology Center, Department of Biochemistry, Stanford University, 3165 Porter Drive, Palo Alto, CA 94304, United States of America.
authors.forename: Ronald W
authors.initials: RW
authors.lastname: Davis
authors.affiliation: Stanford Genome Technology Center, Department of Biochemistry, Stanford University, 3165 Porter Drive, Palo Alto, CA 94304, United States of America.
authors.forename: Laurel D
authors.initials: LD
authors.lastname: Crosby
mesh_terms: D000464:Alginates / Q000737:chemistry*; D002214:Capsules; D002469:Cell Separation / Q000379:methods*; D005240:Feasibility Studies; D016680:Genome, Bacterial / Q000235:genetics*; D020723:Glucuronic Acid / Q000737:chemistry; D006603:Hexuronic Acids / Q000737:chemistry; D008863:Microspheres; D016133:Polymerase Chain Reaction / Q000379:methods*; D012242:Rhodobacter sphaeroides / Q000166:cytology* / Q000235:genetics*; D017422:Sequence Analysis, DNA
chemical_list: D000464:Alginates; D002214:Capsules; D006603:Hexuronic Acids; D020723:Glucuronic Acid