Reconstructing each cell's genome within complex microbial communities-dream or reality?

Abstract

As the vast majority of microorganisms have yet to be cultivated in a laboratory setting, access to their genetic makeup has largely been limited to cultivation-independent methods. These methods, namely metagenomics and more recently single-cell genomics, have become cornerstones for microbial ecology and environmental microbiology. One ultimate goal is the recovery of genome sequences from each cell within an environment to move toward a better understanding of community metabolic potential and to provide substrate for experimental work. As single-cell sequencing has the ability to decipher all sequence information contained in an individual cell, this method holds great promise in tackling such challenge. Methodological limitations and inherent biases however do exist, which will be discussed here based on environmental and benchmark data, to assess how far we are from reaching this goal.

Keywords: environmental microbiology; genome completeness; genome quality; microbial ecology; multiple displacement amplification; single-cell sequencing.

Figure 1

(A) Percentage of single cells sorted from a variety of environmental samples that were successfully amplified by MDA. A minimum of 800 single cells were sorted for each of these sites. (B) Abundance of taxa in 16S rRNA gene tag sequence data as compared to the SAG libraries for four diverse environmental samples. Taxa represent phyla of bacteria and the archaeal class Methanomicrobia.

Figure 2

Benchmark single-cell experiment using three reference strains with finished genomes: P, Pedobacter heparinus; E, Escherichia coli; and M, Meiothermus ruber. (A) Number of assembled contigs >2 kb in length normalized by genome size. (B) Largest assembled contig. (C) The length of the shortest contig among those that collectively cover half of the assembly (L50) normalized by genome size. (D) Number of mismatches between the assembly and the reference genome normalized by genome size. (E) Number of insertions and deletions in the assembly when compared to the reference genome normalized by genome size. (F) Number of misassemblies when compared to the reference genomes normalized by genome size. (G) Percentage of the genome recovered in the assembly when the reads from multiple SAGs are combined together and assembled. (H) Genome recovery vs. real-time MDA crossing point (CP) value.