Bacterial species from natural environments, exhibiting a great degree of genetic diversity that has yet to be characterized, pose a specific challenge to whole genome amplification (WGA) from single cells. A major challenge is establishing an effective, compatible, and controlled lysis protocol. We present a novel lysis protocol that can be used to extract genomic information from a single cyanobacterium of Synechocystis sp. PCC 6803 known to have multilayer cell wall structures that resist conventional lysis methods. Simple but effective strategies for releasing genomic DNA from captured cells while retaining cellular identities for single-cell analysis are presented. Successful sequencing of genetic elements from single-cell amplicons prepared by multiple displacement amplification (MDA) is demonstrated for selected genes (15 loci nearly equally spaced throughout the main chromosome).
Kalisky, T.; Blainey, P.; Quake, S.R. Genomic analysis at the single-cell level. Annu. Rev. Genet. 2011, 45, 431–445, doi:10.1146/annurev-genet-102209-163607.
[3]
Stepanauskas, R. Single cell genomics: An individual look at microbes. Curr. Opin. Microbiol. 2012, 15, 613–620, doi:10.1016/j.mib.2012.09.001.
[4]
Dean, F.B.; Nelson, J.R.; Giesler, T.L.; Lasken, R.S. Rapid amplification of plasmid and phage DNA using Phi29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res. 2001, 11, 1095–1099, doi:10.1101/gr.180501.
[5]
Zhang, K.; Martiny, A.C.; Reppas, N.B.; Barry, K.W.; Malek, J.; Chisholm, S.W.; Church, G.M. Sequencing genomes from single cells by polymerase cloning. Nat. Biotechnol. 2006, 24, 680–686, doi:10.1038/nbt1214.
[6]
Woyke, T.; Xie, G.; Copeland, A.; González, J.M.; Han, C.; Kiss, H.; Saw, J.H.; Senin, P.; Yang, C.; Chatterji, S.; et al. Assembling the marine metagenome, one cell at a time. PLoS One 2009, 4, e5299, doi:10.1371/journal.pone.0005299.
[7]
Woyke, T.; Tighe, D.; Mavromatis, K.; Clum, A.; Copeland, A.; Schackwitz, W.; Lapidus, A.; Wu, D.; McCutcheon, J.P.; McDonald, B.R.; et al. One bacterial cell, one complete genome. PLoS One 2010, 5, e10314, doi:10.1371/journal.pone.0010314.
[8]
Arakaki, A.; Shibusawa, M.; Hosokawa, M.; Matsunaga, T. Preparation of genomic DNA from a single species of uncultured magnetotactic bacterium by multiple-displacement amplification. Appl. Environ. Microbiol. 2010, 76, 1480–1485, doi:10.1128/AEM.02124-09.
[9]
Zong, C.; Lu, S.; Chapman, A.R.; Xie, X.S. Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. Science 2012, 338, 1622–1626, doi:10.1126/science.1229164.
Marcy, Y.; Ouverney, C.; Bik, E.M.; L？sekann, T.; Ivanova, N.; Martin, H.G.; Szeto, E.; Platt, D.; Hugenholtz, P.; Relman, D.A.; et al. Dissecting biological “dark matter” with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth. Proc. Natl. Acad. Sci. USA 2007, 104, 11889–11894, doi:10.1073/pnas.0704662104.
[12]
Blainey, P.C.; Mosier, A.C.; Potanina, A.; Francis, C.A.; Quake, S.R. Genome of a low-salinity ammonia-oxidizing archaeon determined by single-cell and metagenomic analysis. PLoS One 2011, 6, e16626, doi:10.1371/journal.pone.0016626.
[13]
Wang, J.; Fan, H.C.; Behr, B.; Quake, S.R. Genome-wide single-cell analysis of recombination activity and de novo mutation rates in human sperm. Cell 2012, 150, 402–412, doi:10.1016/j.cell.2012.06.030.
[14]
Kaneko, T.; Sato, S.; Kotani, H.; Tanaka, A.; Asamizu, E.; Nakamura, Y.; Miyajima, N.; Hirosawa, M.; Sugiura, M.; Sasamoto, S.; et al. Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res. 1996, 3, 109–136, doi:10.1093/dnares/3.3.109.
[15]
Hoiczyk, E.; Hansel, A. Cyanobacterial cell walls: News from an unusual prokaryotic envelope. J. Bacteriol. 2000, 182, 1191–1199, doi:10.1128/JB.182.5.1191-1199.2000.
[16]
Liu, X.; Curtiss, R. Nickel-inducible lysis system in Synechocystis sp. PCC 6803. Proc. Natl. Acad. Sci. USA 2009, 106, 21550–21554, doi:10.1073/pnas.0911953106.
[17]
Blainey, P.C.; Quake, S.R. Digital MDA for enumeration of total nucleic acid contamination. Nucleic Acids Res. 2011, 39, e19, doi:10.1093/nar/gkq1074.
[18]
Chueh, B.; Li, C.-W.; Wu, H.; Davison, M.; Wei, H.; Bhaya, D.; Zare, R.N. Whole gene amplification and protein separation from a few cells. Anal. Biochem. 2011, 411, 64–70, doi:10.1016/j.ab.2010.12.028.
[19]
Ye, J.; Coulouris, G.; Zaretskaya, I.; Cutcutache, I.; Rozen, S.; Madden, T.L. Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinforma. 2012, 13, 134, doi:10.1186/1471-2105-13-134.
[20]
Woyke, T.; Sczyrba, A.; Lee, J.; Rinke, C.; Tighe, D.; Clingenpeel, S.; Malmstrom, R.; Stepanauskas, R.; Cheng, J.-F. Decontamination of MDA reagents for single cell whole genome amplification. PLoS One 2011, 6, e26161, doi:10.1371/journal.pone.0026161.
[21]
Wu, X.; Zarka, A.; Boussiba, S. A simplified protocol for preparing DNA from filamentous cyanobacteria. Plant Mol. Biol. Report. 2000, 18, 385–392, doi:10.1007/BF02825067.
[22]
Hall, E.W. Microfluidic platforms for single-cell analyses. Ph.D. Thesis, Stanford University, Stanford, CA, USA, October 2012.
