Specific single-cell isolation and genomic amplification of uncultured microorganisms

Abstract

We in this study describe a new method for genomic studies of individual uncultured prokaryotic organisms, which was used for the isolation and partial genome sequencing of a soil archaeon. The diversity of Archaea in a soil sample was mapped by generating a clone library using group-specific primers in combination with a terminal restriction fragment length polymorphism profile. Intact cells were extracted from the environmental sample, and fluorescent in situ hybridization probing with Cy3-labeled probes designed from the clone library was subsequently used to detect the organisms of interest. Single cells with a bright fluorescent signal were isolated using a micromanipulator and the genome of the single isolated cells served as a template for multiple displacement amplification (MDA) using the Phi29 DNA polymerase. The generated MDA product was afterwards used for 16S rRNA gene sequence analysis and shotgun-cloned for additional genomic analysis. Sequence analysis showed >99% 16S rRNA gene homology to soil crenarchaeotal clone SCA1170 and shotgun fragments had the closest match to a crenarchaeotal BAC clone previously retrieved from a soil sample. The system was validated using Methanothermobacter thermoautotrophicus as single-cell test organism, and the validation setup produced 100% sequence homology to the ten tested regions of the genome of this organism.

Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology

“…Micromanipulation is relatively time consuming but is a powerful research tool allowing observation of cell morphology, repeated washing in rinse buffers to remove contaminating free DNA and visual documentation that a single cell was captured and delivered to the MDA reaction (Ishøy et al, 2006;Kvist et al, 2007). As discussed below, the isolation of single or a few cells for use in MDA reactions has also been carried out by fluorescence-activated cell sorting (Raghunathan et al, 2004;Podar et al, 2007;Stepanauskas and Sieracki, 2007), microfluidics (Marcy et al, 2007a, b) or micromanipulation (Kvist et al, 2007). Cell sorting by FISH labelling of 16S rRNA followed by automated cell lysis and MDA using liquid-handling stations is expected to provide unprecedented high throughput for the analysis of uncultivated cells.…”

Coupling FACS and Genomic Methods for the Characterization of Uncultivated Symbionts

“…Other methods such as serial dilution (Zhang, et al, 2006), micromanipulation (Kvist, et al, 2007;Hongoh, et al, 2 2008), laser capture microdissection (Navin, et al, 2011), Raman tweezers (Brehm-Stecher & Johnson, 2004;Huang, et al, 2009) and microfluidics (Marcy, et al, 2007;Blainey, et al, 2011) have also been used effectively to separate cells prior to genetic or genomic analysis. However, for characterizing uncultivated symbionts these methods fall short of FACS in a number of ways.…”

Contemporary molecular tools in microbial ecology and their application to advancing biotechnology

“…The advent of single cell genomics was indeed a great leap in studying (1) (Brosius, Palmer et al 1978); (2) (Carbon, Ebel et al 1981); (3) (Lane, Pace et al 1985); (4) (Weller and Ward 1989); (5) (Schmidt, DeLong et al 1991); (6) (Healy, Ray et al 1995); (7) (Handelsman, Rondon et al 1998); (8) , Rondon, August et al 2000; (9) (Breitbart, Salamon et al 2002); (10) (Tyson, Chapman et al 2004); (11) (Margulies, Egholm et al 2005); (12) (Kvist, Ahring et al 2007, Marcy, Ouverney et al 2007); (13) (Bentley, Balasubramanian et al 2008, Harris, Buzby et al 2008, Valouev, Ichikawa et al 2008); (14) (Rodrigue, Malmstrom et al 2009); (15) (Yooseph, Nealson et al 2010); (16) (Siegl, Kamke et al 2011, Youssef, Blainey et al 2011); (17) (Freeman, Gurgui et al 2012); (18) (Rinke, Schwientek et al 2013); (19) (Chi 2014). (Wilson, Mori et al 2014) …”

“…Also, taxonomic assignments of metagenomic reads/contigs remain challenging. The recent breakthrough of single-cell genome sequencing (SCGC), in which sequencing the genomic material of a single physically separated uncultured microbial cell is possible, alleviated many bottlenecks in metagenomics (Kvist, Ahring et al 2007, Marcy, Ouverney et al 2007. In this technology, DNA from a single cell is isolated and amplified through multiple displacement amplification (MDA) in a sufficient quantity to be sequenced by the current day genome sequencers.…”

“…The first application of MDA and single-cell genomics in the field of microbial ecology was the publication of a partial genome sequence of an uncultured archaeon from a soil sample (Kvist, Ahring et al 2007). Subsequently, the genomes of cells of the rare and uncultivated candidate phylum TM7 were published from two ecological niches, one from a human mouth sample (Marcy, Ouverney et al 2007), and the other from a soil sample (Podar, Abulencia et al 2007).…”