One bacterial cell, one complete genome

Abstract

While the bulk of the finished microbial genomes sequenced to date are derived from cultured bacterial and archaeal representatives, the vast majority of microorganisms elude current culturing attempts, severely limiting the ability to recover complete or even partial genomes from these environmental species. Single cell genomics is a novel culture-independent approach, which enables access to the genetic material of an individual cell. No single cell genome has to our knowledge been closed and finished to date. Here we report the completed genome from an uncultured single cell of Candidatus Sulcia muelleri DMIN. Digital PCR on single symbiont cells isolated from the bacteriome of the green sharpshooter Draeculacephala minerva bacteriome allowed us to assess that this bacteria is polyploid with genome copies ranging from approximately 200-900 per cell, making it a most suitable target for single cell finishing efforts. For single cell shotgun sequencing, an individual Sulcia cell was isolated and whole genome amplified by multiple displacement amplification (MDA). Sanger-based finishing methods allowed us to close the genome. To verify the correctness of our single cell genome and exclude MDA-derived artifacts, we independently shotgun sequenced and assembled the Sulcia genome from pooled bacteriomes using a metagenomic approach, yielding a nearly identical genome. Four variations we detected appear to be genuine biological differences between the two samples. Comparison of the single cell genome with bacteriome metagenomic sequence data detected two single nucleotide polymorphisms (SNPs), indicating extremely low genetic diversity within a Sulcia population. This study demonstrates the power of single cell genomics to generate a complete, high quality, non-composite reference genome within an environmental sample, which can be used for population genetic analyzes.

Figure 1. Sulcia cell isolation and sequence coverage, closure and polishing locations along the Sulcia DMIN single cell genome.

(A) Micromanipulation of the single Sulcia cell from the sharpshooter bacteriome metasample. (B) Sequence coverage including closure and polishing locations along the finished, circular Sulcia DMIN genome with circles corresponding to following features, starting with outermost circle: (1) Illumina sequence coverage ranging from 0–3276 (mean 303+−386), (2) pyrosequence sequence coverage ranging from 0–231 (mean 42+−39), (3) Sanger sequence coverage ranging from 0–30 (mean 10+−7), (4) locations of captured (green) and uncaptured gaps (orange), (5) polishing locations corrected using Illumina (blue) and Sanger (purple) seqeunce, (6) GC content heat map (dark blue to light green = low to high values) and (7) GC skew.

Figure 2. Distribution of protein similarities between the two available genomes of Candidatus Sulcia muelleri from sharpshooters.