title: Droplet-based pyrosequencing using digital microfluidics.
abstract: The feasibility of implementing pyrosequencing chemistry within droplets using electrowetting-based digital microfluidics is reported. An array of electrodes patterned on a printed-circuit board was used to control the formation, transportation, merging, mixing, and splitting of submicroliter-sized droplets contained within an oil-filled chamber. A three-enzyme pyrosequencing protocol was implemented in which individual droplets contained enzymes, deoxyribonucleotide triphosphates (dNTPs), and DNA templates. The DNA templates were anchored to magnetic beads which enabled them to be thoroughly washed between nucleotide additions. Reagents and protocols were optimized to maximize signal over background, linearity of response, cycle efficiency, and wash efficiency. As an initial demonstration of feasibility, a portion of a 229 bp  Candida parapsilosis  template was sequenced using both a de novo protocol and a resequencing protocol. The resequencing protocol generated over 60 bp of sequence with 100% sequence accuracy based on raw pyrogram levels. Excellent linearity was observed for all of the homopolymers (two, three, or four nucleotides) contained in the  C. parapsilosis  sequence. With improvements in microfluidic design it is expected that longer reads, higher throughput, and improved process integration (i.e., "sample-to-sequence" capability) could eventually be achieved using this low-cost platform.
authors.affiliation: Advanced Liquid Logic Incorporated, Research Triangle Park, North Carolina, United States.
authors.forename: Deborah J
authors.initials: DJ
authors.lastname: Boles
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: Benton
authors.forename: Germaine J
authors.initials: GJ
authors.lastname: Siew
authors.forename: Miriam H
authors.initials: MH
authors.lastname: Levy
authors.forename: Prasanna K
authors.initials: PK
authors.lastname: Thwar
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Sandahl
authors.forename: Jeremy L
authors.initials: JL
authors.lastname: Rouse
authors.forename: Lisa C
authors.initials: LC
authors.lastname: Perkins
authors.forename: Arjun P
authors.initials: AP
authors.lastname: Sudarsan
authors.forename: Roxana
authors.initials: R
authors.lastname: Jalili
authors.forename: Vamsee K
authors.initials: VK
authors.lastname: Pamula
authors.forename: Vijay
authors.initials: V
authors.lastname: Srinivasan
authors.forename: Richard B
authors.initials: RB
authors.lastname: Fair
authors.forename: Peter B
authors.initials: PB
authors.lastname: Griffin
authors.forename: Allen E
authors.initials: AE
authors.lastname: Eckhardt
authors.forename: Michael G
authors.initials: MG
authors.lastname: Pollack
mesh_terms: D001483:Base Sequence; D002175:Candida / Q000235:genetics; D004271:DNA, Fungal / Q000032:analysis* / Q000235:genetics*; D003854:Deoxyribonucleotides / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D004566:Electrodes; D004798:Enzymes / Q000737:chemistry / Q000378:metabolism; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*; D013698:Templates, Genetic
chemical_list: D004271:DNA, Fungal; D003854:Deoxyribonucleotides; D004798:Enzymes