Capturing and cultivating single bacterial cells in gel microdroplets to obtain near-complete genomes

Nature Protocols

Volumn 9, Issue 3, 2014, Pages 608-621

  Dichosa, Armand E K ^a   Daughton, Ashlynn R ^a   Reitenga, Krista G ^a   Fitzsimons, Michael S ^a,b   Han, Cliff S ^a  

^a LOS ALAMOS NATIONAL LABORATORY   (United States)

^b NuGEN Technologies   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BACTERIAL DNA; DNA 16S;

ARTICLE; BACILLUS SUBTILIS; BACTERIAL CELL; BACTERIAL GENOME; BACTERIAL GROWTH; BACTERIUM COLONY; BACTERIUM CULTURE; CELL ISOLATION; COCULTURE; CONTROLLED STUDY; ESCHERICHIA COLI; FLOW CYTOMETRY; GEL; GEL MICRODROPLET; GENE SEQUENCE; GENOME ANALYSIS; IN VITRO STUDY; NONHUMAN; PRIORITY JOURNAL; PROCESS DEVELOPMENT; SINGLE CELL ANALYSIS;

BACTERIA; FLOW CYTOMETRY; GASTROINTESTINAL TRACT; GELS; GENOME, BACTERIAL; GENOMICS; HUMANS; MOUTH; SINGLE-CELL ANALYSIS;

EID: 84895482501     PISSN: 17542189     EISSN: 17502799     Source Type: Journal    
DOI: 10.1038/nprot.2014.034     Document Type: Article

References (36)

1. Lasken, R. Single-cell genomic sequencing using multiple displacement amplification. Curr. Opin. Microbiol. 10, 510-516 (2007
2. Marcy, Y. 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 104, 11889-11894 (2007
3. Podar, M. et al. Targeted access to the genomes of low-Abundance organisms in complex microbial communities. Appl. Environ. Microbiol. 73, 3205-3214 (2007
4. Stepanauskas, R. & Sieracki, M.E. Matching phylogeny and metabolism in the uncultured marine bacteria, one cell at a time. Proc. Natl. Acad. Sci. USA 104, 9052-9057 (2007
5. Rodrigue, S. et al. Whole genome amplification and de novo assembly of single bacterial cells. PLoS ONE 4, e6864 (2009
6. Woyke, T. et al. Assembling the marine metagenome, one cell at a time. PLoS ONE 4, e5299 (2009
7. Rinke, C. et al. Insights into the phylogeny and coding potential of microbial dark matter. Nature 499, 431-437 (2013
8. Ballantyne, K.N., Oorschot, R.A., Muharamc, I., Daal, A. & Mitchell, R.J. Decreasing amplification bias associated with multiple displacement amplification and short tandem repeat genotyping. Anal. Biochem. 368, 222-229 (2007
9. Pan, X. et al. A procedure for highly specific, sensitive, and unbiased whole-genome amplification. Proc. Natl. Acad. Sci. USA 105, 15499-15504 (2008
10. Lasken, R.S. & Stockwell, T.B. Mechanism of chimera formation during the multiple displacement amplification reaction. BMC Biotechnol. 7, 1-11 (2007
11. Fitzsimons, M.S. et al. Nearly finished genomes produced using gel microdroplet culturing reveals substantial intraspecies diversity within the human microbiome. Genome Res. 23, 878-888 (2013
12. Seth-Smith, H.M.B. et al. Whole-genome sequences of Chlamydia trachomatis directly from clinical samples without culture. Genome Res. 23, 855-866 (2013
13. Blainey, P.C. & Quake, S.R. Digital MDA for enumeration of total nucleic acid contamination. Nucleic Acids Res. 39, e19 (2010
14. Woyke, T. et al. Decontamination of MDA reagents for single cell whole genome amplification. PLoS ONE 6, e26161 (2011
15. Dichosa, A.E.K. et al. Artificial polyploidy improves bacterial single cell genome recovery. PLoS ONE 7, e37387 (2012
16. Woyke, T. et al. One bacterial cell, one complete genome. PLoS ONE 5, e10314 (2010
17. Seth-Smith, H.M.B. et al. Generating whole bacterial genome sequences of low-Abundance species from complex samples with IMS-MDA. Nat. Protoc. 8, 2404-2412 (2013
18. McLean, J.S. et al. Candidate phylum TM6 genome recovered from a hospital sink biofilm provides genomic insights into this uncultivated phylum. Proc. Natl. Acad. Sci. USA 110, E2390-E2399 (2013
19. Weaver, J.C., Williams, G.B., Klibanov, A. & Demain, A.L. Gel microdroplets: Rapid detection and enumeration of individual microorganisms by their metabolic activity. Nat. Biotechnol. 6, 1084-1089 (1988
20. Williams, G.B., Weaver, J.C. & Demain, A.L. Rapid microbial detection and enumeration using gel microdroplets and colorimetric or fluorescence indicator systems. J. Clin. Microbiol. 28, 1002-1008 (1990
21. Borth, N., Zeyda, M. & Katinge, H. Efficient selection of high-producing subclones during gene amplification of recombinant Chinese hamster ovary cells by flow cytometry and cell sorting. Biotechnol. Bioeng. 71, 266-273 (2000
22. Katsuragi, T., Tanaka, S., Nagahiro, S. & Tani, Y. Gel microdroplet technique leaving microorganisms alive for sorting by flow cytometry. J. Microbiol. Methods 42, 81-86 (2000
23. Manome, A. et al. Application of gel microdroplet and flow cytometry techniques to selective enrichment of non-growing bacterial cells. FEMS Microbiol. Lett. 197, 29-33 (2001
24. Cox, D.L., Akins, D.R., Porcella, S.F., Norgard, M.V. & Radolf, J.D. Treponema pallidum in gel microdroplets: A novel strategy for investigation of treponemal molecular architecture. Mol. Microbiol. 15, 1151-1164 (1995
25. Cox, D.L. et al. Limited surface exposure of Borrelia burgdorferi outer surface lipoproteins. Proc. Natl. Acad. Sci. USA 93, 7973-7978 (1996
26. Ryan, C., Nguyen, B-T. & Sullivan, S.J. Rapid assay for mycobacterial growth and antibiotic susceptibility using gel microdrop encapsulation. J. Clin. Microbiol. 33, 1720-1726 (1995
27. Powell, K.T. & Weaver, J.C. Gel microdroplets and flow cytometry: Rapid determination of antibody secretion by individual cells within a cell population. Nat. Biotechnol. 8, 333-337 (1990
28. Zengler, K. et al. Cultivating the uncultured. Proc. Natl. Acad. Sci. USA 99, 15681-15686 (2002
29. One Cell Systems, Inc. Gel microdrop assay systems, GMD bacterial growth assay: Experimental guidelines and protocols. Doc. no. 9005. version 004133 (2003
30. Edwards, U., Rogall, T., Blöcker, H., Emde, M. & Böttger, E.C. Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res. 17, 7843-7853 (1989
31. Turner, S., Pryer, K.M., Miao, V.P.W. & Palmer, J.D. Investigating deep phylogenetic relationships among cyanobacteria and plastids by small subunit rRNA sequence analysis. J. Eukaryotic Microbiol. 46, 327-338 (1999
32. Wilson, K.H., Blitchington, R.B. & Greene, R.C. Amplification of bacterial 16S ribosomal DNA with polymerase chain reaction. J. Clin. Microbiol. 28, 1942-1946 (1990
33. Baker, G.C. & Cowan, D.A. 16 S rDNA primers and the unbiased assessment of thermophile diversity. Biochem. Soc. Transac. 32, 218-221 (2004
34. Berney, M., Hammes, F. & Bosshard, F. Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight kit in combination with flow cytometry. Appl. Environ. Microbiol. 73, 3283-3290 (2007
35. Gasol, J.M. & Giorgio, P.A.d. Using flow cytometry for counting natural planktonic bacteria and understanding the structure of planktonic bacterial communities. Scientia Marina 64, 197-224 (2000
36. Hammes, F. et al. Flow-cytometric total bacterial cell counts as a descriptive microbiological parameter for drinking water treatment processes. Water Res. 42, 269-277 (2008