title: Challenges of metagenomics and single-cell genomics approaches for exploring cyanobacterial diversity.
abstract: Cyanobacteria have played a crucial role in the history of early earth and continue to be instrumental in shaping our planet, yet applications of cutting edge technology have not yet been widely used to explore cyanobacterial diversity. To provide adequate background, we briefly review current sequencing technologies and their innovative uses in genomics and metagenomics. Next, we focus on current cell capture technologies and the challenges of using them with cyanobacteria. We illustrate the utility in coupling breakthroughs in DNA amplification with cell capture platforms, with an example of microfluidic isolation and subsequent targeted amplicon sequencing from individual terrestrial thermophilic cyanobacteria. Single cells of thermophilic, unicellular Synechococcus sp. JA-2-3-B'a(2-13) (Syn OS-B') were sorted in a microfluidic device, lysed, and subjected to whole genome amplification by multiple displacement amplification. We amplified regions from specific CRISPR spacer arrays, which are known to be highly diverse, contain semi-palindromic repeats which form secondary structure, and can be difficult to amplify. Cell capture, lysis, and genome amplification on a microfluidic device have been optimized, setting a stage for further investigations of individual cyanobacterial cells isolated directly from natural populations.
authors.affiliation: Department of Plant Biology, Carnegie Institution of Science, 260 Panama Street, Stanford, CA, 94305, USA. shellblu@stanford.edu.
authors.forename: Michelle
authors.initials: M
authors.lastname: Davison
authors.affiliation: SRI International, 333 Ravenswood Ave, Menlo Park, CA, 94025, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Hall
authors.affiliation: Department of Chemistry, Stanford University, 333 Campus Drive Mudd Building, Room 121, Stanford, CA, 94305-4401, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Zare
authors.affiliation: Department of Plant Biology, Carnegie Institution of Science, 260 Panama Street, Stanford, CA, 94305, USA.
authors.forename: Devaki
authors.initials: D
authors.lastname: Bhaya
mesh_terms: D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D000458:Cyanobacteria / Q000235:genetics*; D014644:Genetic Variation; D023281:Genomics / Q000379:methods*; D056656:Lab-On-A-Chip Devices; D056186:Metagenomics / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods; D059010:Single-Cell Analysis / Q000295:instrumentation / Q000379:methods*; D046940:Synechococcus / Q000235:genetics
keywords: CRISPR; Cyanobacteria; Microfluidics; Multiple displacement amplification (MDA); Single cell; Whole genome amplification (WGA)