Abstract
Single-cell genomics allows bypassing the culturing step and to directly access environmental microbes one cell at a time. The method has been successfully applied to explore archaeal and bacterial candidate phyla, referred to as microbial dark matter. Here I summarize the single-cell genomics workflow, including sample preparation and preservation, high-throughput fluorescence-activated cell sorting, cell lysis and amplification of environmental samples. Furthermore I describe phylogenetic screening based on 16S rRNA genes and suggest a suitable library preparation and sequencing approach.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169
Dodsworth JA, Blainey PC, Murugapiran SK, Swingley WD, Ross CA, Tringe SG, Chain PSG, Scholz MB, Lo C-C, Raymond J, Quake SR, Hedlund BP (2013) Single-cell and metagenomic analyses indicate a fermentative and saccharolytic lifestyle for members of the OP9 lineage. Nat Commun 4:1854. https://doi.org/10.1038/ncomms2884
Kamke J, Sczyrba A, Ivanova N, Schwientek P, Rinke C, Mavromatis K, Woyke T, Hentschel U (2013) Single-cell genomics reveals complex carbohydrate degradation patterns in poribacterial symbionts of marine sponges. ISME J 7(12):2287–2300. https://doi.org/10.1038/ismej.2013.111
Campbell JH, O’Donoghue P, Campbell AG, Schwientek P, Sczyrba A, Woyke T, Söll D, Podar M (2013) UGA is an additional glycine codon in uncultured SR1 bacteria from the human microbiota. Proc Natl Acad Sci 110:5540–5545. https://doi.org/10.1073/pnas.1303090110
McLean JS, Lombardo M-J, Badger JH, Edlund A, Novotny M, Yee-Greenbaum J, Vyahhi N, Hall AP, Yang Y, Dupont CL, Ziegler MG, Chitsaz H, Allen AE, Yooseph S, Tesler G, Pevzner PA, Friedman RM, Nealson KH, Venter JC, Lasken RS (2013) Candidate phylum TM6 genome recovered from a hospital sink biofilm provides genomic insights into this uncultivated phylum. Proc Natl Acad Sci 110(26):E2390–E2399. https://doi.org/10.1073/pnas.1219809110
Marcy Y, Ouverney C, Bik EM, Lösekann T, Ivanova N, Martin HG, Szeto E, Platt D, Hugenholtz P, Relman DA, Quake SR (2007) Dissecting biological “dark matter” with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth. Proc Natl Acad Sci 104:11889–11894. https://doi.org/10.1073/pnas.0704662104
Rinke C, Schwientek P, Sczyrba A, Ivanova NN, Anderson IJ, Cheng J-F, Darling A, Malfatti S, Swan BK, Gies EA, Dodsworth JA, Hedlund BP, Tsiamis G, Sievert SM, Liu W-T, Eisen JA, Hallam SJ, Kyrpides NC, Stepanauskas R, Rubin EM, Hugenholtz P, Woyke T (2013) Insights into the phylogeny and coding potential of microbial dark matter. Nature 499:431–437. https://doi.org/10.1038/nature12352
Kashtan N, Roggensack SE, Rodrigue S, Thompson JW, Biller SJ, Coe A, Ding H, Marttinen P, Malmstrom RR, Stocker R, Follows MJ, Stepanauskas R, Chisholm SW (2014) Single-cell genomics reveals hundreds of coexisting subpopulations in wild Prochlorococcus. Science 344:416–420. https://doi.org/10.1126/science.1248575
Yoon HS, Price DC, Stepanauskas R, Rajah VD, Sieracki ME, Wilson WH, Yang EC, Duffy S, Bhattacharya D (2011) Single-cell genomics reveals organismal interactions in uncultivated marine protists. Science 332:714–717. https://doi.org/10.1126/science.1203163
Woyke T, Tighe D, Mavromatis K, Clum A, Copeland A, Schackwitz W, Lapidus A, Wu D, McCutcheon JP, McDonald BR, Moran NA, Bristow J, Cheng J-F (2010) One bacterial cell, one complete genome. PLoS One 5:e10314. https://doi.org/10.1371/journal.pone.0010314
Landry ZC, Giovanonni SJ, Quake SR, Blainey PC (2013) Chapter 4: Optofluidic cell selection from complex microbial communities for single-genome analysis. In: DeLong EF (ed) Methods Enzymol. Academic Press, Cambridge, pp 61–90
Frumkin D, Wasserstrom A, Itzkovitz S, Harmelin A, Rechavi G, Shapiro E (2008) Amplification of multiple genomic loci from single cells isolated by laser micro-dissection of tissues. BMC Biotechnol 8:17. https://doi.org/10.1186/1472-6750-8-17
Rinke C, Lee J, Nath N, Goudeau D, Thompson B, Poulton N, Dmitrieff E, Malmstrom R, Stepanauskas R, Woyke T (2014) Obtaining genomes from uncultivated environmental microorganisms using FACS–based single-cell genomics. Nat Protoc 9:1038–1048
Clingenpeel S, Clum A, Schwientek P, Rinke C, Woyke T (2014) Reconstructing each cell’s genome within complex microbial communities - dream or reality? Microb Physiol Metab 5:771. https://doi.org/10.3389/fmicb.2014.00771
Zong C, Lu S, Chapman AR, Xie XS (2012) Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. Science 338:1622–1626. https://doi.org/10.1126/science.1229164
de Bourcy CFA, De Vlaminck I, Kanbar JN, Wang J, Gawad C, Quake SR (2014) A quantitative comparison of single-cell whole genome amplification methods. PLoS One 9:e105585. https://doi.org/10.1371/journal.pone.0105585
Rinke C, Serene L, Ben W, Jean-Baptiste R, Adam S, Xuyen L, Margaret KB, Stocker R, Seymour J, Tyson GW, Hugenholtz P (2016) Validation of picogram-input DNA libraries for microscale metagenomic. PeerJ 4:e2486. https://doi.org/10.7717/peerj.2486
Thomsen PF, Willerslev E (2015) Environmental DNA—an emerging tool in conservation for monitoring past and present biodiversity. Biol Conserv 183:4–18. https://doi.org/10.1016/j.biocon.2014.11.019
Acknowledgments
The work was conducted at the Australian Centre for Ecogenomics (ACE) at UQ, and was supported by the ARC Discovery Project DP160103811. I would like to thank the ACE team for support, especially Dr. Michael Nefedov and Alexander Baker.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Rinke, C. (2018). Single-Cell Genomics of Microbial Dark Matter. In: Beiko, R., Hsiao, W., Parkinson, J. (eds) Microbiome Analysis. Methods in Molecular Biology, vol 1849. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8728-3_7
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8728-3_7
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8726-9
Online ISBN: 978-1-4939-8728-3
eBook Packages: Springer Protocols