Single cell genome sequencing
Highlights
► Need and impact of single cell genomics on understanding microbial communities. ► Highlights the latest tools for single cell isolation, lysis, and amplification. ► Comments on remaining challenges on successful single cell sequencing efforts.
Introduction
Microbes, the most abundant species on earth, play an important role in ecological processes in making, breaking down, and recycling the essential chemicals of life. Microbes are also related to our health and are abundant in our bodies — we have 10 times as many microbes living on and inside us as human cells. Despite their importance, it is estimated that 90–99% of microbes have not been characterized because they cannot be cultured in a laboratory. Hence, culture-independent techniques such as fluorescence in situ hybridization (FISH), PCR, microarrays, and sequencing of the 16S rRNA gene are relied upon to detect and analyze microbes. More recently, the advent of next-generation sequencing has allowed large-scale shotgun sequencing of collective genomes in a microbial community. This has allowed an unprecedented access to uncultured microbial communities and their activities and has been applied to a wide variety of habitats ranging from termite gut to marine environments [1, 2]. However, a major drawback with the shotgun sequencing of the metagenome is that while it provides information on the species present and the function(s) of the community, it cannot link the function back to the species. Moreover, in the majority of cases, metagenomic sequencing does not allow assembly of individual genomes in the community.
To overcome the problems associated with metagenomics, research efforts have generally focused on the sequencing of individual cells via the employment of whole genome amplification strategies [3]. Single cell sequencing has been applied to numerous environmental microbes (prokaryotic and eukaryotic microorganisms as well as environmental viruses) including T7 from human mouth and soil, Flavobacteria and heterotrophic protists from ocean, Termite Group 1 and Desulfovibrio from termite gut, Leptothrix from iron mats, Sulcia from a green sharpshooter, ammonia oxidizing archaea from a low-salinity estuary, and Poribacteria from marine sponge [3, 4••, 5, 6, 7, 8, 9, 10•, 11, 12••]. Single cell genome sequencing involves the isolation of single cells from the environmental sample, cell lysis and multiple displacement amplification (MDA) followed by whole genome sequencing and analysis as depicted in Figure 1 and described below.
Section snippets
Single cell isolation
The first step in single cell genomics is the isolation of individual cells from microbial communities. Several single cell isolation methods have been developed including serial dilution, micromanipulation, laser capture microdissection, Raman tweezers, fluorescence activated cell sorting (FACS), and microfluidics. Serial dilution, a simple and inexpensive technique mostly used for culturing studies, has been used to isolate single cells of Escherichia coli and Prochlorococcus marinus for
Cell lysis and gDNA extraction
After isolating single cells, the next step is to lyse them to extract genomic DNA (gDNA). This is perhaps the most critical step as the success of subsequent whole genome amplification depends on the availability and quality of gDNA, especially for small prokaryotic cells that contain only a few femtograms of DNA. The lysis method should be harsh enough to lyse the cells while gentle enough to preserve the integrity of gDNA. Of the several lysis methods available, none exists that can handle
Whole genome amplification
Next-generation sequencing technologies require micrograms of DNA and hence, amplification is a crucial step for sequencing of cells typically containing femtograms of DNA. MDA has become the method of choice for whole genome amplification from single cells. It is an isothermal amplification method that uses random primers and Phi29 DNA polymerase for generating fairly large fragments (10–20 kb) with high fidelity [31]. Although it is proven to be the best method for whole genome amplification
Analysis of single cell sequence data
The earlier studies utilizing Sanger sequencing [8, 13] or short-read length 454 pyrosequencing [7] resulted in partial recovery of SAGs. While Zhang et al. were able to span 66% of Prochlorococcus genome with 7.2 Mb of high-quality Sanger reads [13], Marcy et al. recovered an undetermined % of the genome in a fragmented assembly of 1825 scaffolds (∼2.86 Mb) by pyrosequencing of three individual cells of uncultured TM7 from the human mouth [7].
The higher throughput and longer reads of
Conclusions
Despite tremendous sequencing efforts, it has not been possible to achieve complete assembly and metabolic reconstruction of individual genomes using metagenomics, except in very simple communities such as in acid mine drainage [44]. With the recent advances in whole genome amplification strategies and sequencing technologies, single cell genomics has complemented metagenomics in unraveling the individual genomes and making it possible to complete genome assembly of novel uncultivated
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
Financial support for preparation and some of the work included was provided by the grants: R01 DE020891, funded by the NIDCR; P50GM085273 (the New Mexico Spatiotemporal Modeling Center) funded by the NIGMS; and ENIGMA, a Lawrence Berkeley National Laboratory Scientific Focus Area Program supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research. Sandia is a multi program laboratory operated by Sandia Corp., a Lockheed Martin Co., for the
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