Deciphering the world around us – the “microbial dark matter” (1) – is a remaining challenge in environmental microbiology. Even after decades of 16S rRNA gene-based surveys, there are strong indications that the diversity of bacteria is still not exhaustively described even on high taxonomic (phylum) levels and that 16S rRNA sequences, representing novel bacterial phyla, hide in public databases and have not yet been detected (2).
Metagenomics and microbial single cell genomics emerged in the past few years due to advancements in whole genome amplification (WGA), next-generation sequencing and bioinformatics. The amount of publicly available metagenomic data is continuously growing. Various portals such as IMG/MG, EBI metagenomics, iMicrobe or MG-RAST contain thousands of metagenome datasets: IMG/M received more than 3000 new metagenomes in 2015 and now includes more than 8000 metagenome datasets in their database (3). EBI metagenomics has more than 5000 metagenomes and almost 900 metatranscriptomes (4). The amount of data available in these databases is steadily increasing. An average of 3 to 4 terabasepairs are added to MG-RAST monthly. This amount is double what it was 10 months ago (5). These huge amounts of data are an incredibly valuable source for uncovering potential new bacterial taxa.
Combining metagenomic data mining with single cell genomics is even more powerful when applied synergistically. As described in a recently published study, Eloe-Fadrosh et al. were able to discover and describe a novel bacterial candidate phylum (“Candidatus Kryptonia”) by analyzing metagenomic data from IMG/M combined with single cell genomics from samples collected from geothermal springs (6). Starting with a collection of 31955 long assembled contigs (≥100 kbp) from 4290 metagenomic data sets (IMG/M), 744 contigs with non-redundant SSU rRNA (small-subunit ribosomal RNA) were further selected, aligned and placed on a reference phylogenetic tree. This led finally to the identification of a distinct lineage and near-complete recovery of 4 distinct genomes. Comparing SSU rRNA primer sets typically used for amplicon sequencing studies with the novel sequences indicated mismatches, which combined with a relatively specific ecological niche could be the reason why this lineage has not been detected yet in previous microbial surveys.
In addition to metagenomics data mining, single cell sampling from 4 geothermal springs was performed. Single cells from the samples were isolated using FACS sorting, followed by WGA with the REPLI-g® Single Cell Kit, PCR screening for genomes from single cells matching “Ca. Kryptonia” SSU rRNA sequences and sequenced on Illumina® MiSeq®. Eighteen single cell genomes with an average genome completeness of 67% were recovered. The high-quality draft genomes from metagenomic data analysis and single cell genomics enabled further analyses in bacteria-virus interaction and the detection of a novel fusion between two different CRISPR-Cas types, which represents the first description of a type I-B/type III-A CRISPR-Cas fusion.
QIAGEN’s REPLI-g Single Cell Kit is used in this study for WGA of isolated single bacterial cells. The optimized multiple displacement technology enables sequence-independent uniform coverage across the genome, for better genome assembly. The single cell optimized chemistry allows starting directly from a bacterial cell. Buffers and reagents undergo a controlled decontamination procedure ensuring the elimination of detectable residual DNA contamination which makes the REPLI-g Single Cell Kit perfectly suited for sensitive applications like microbial single cell genomics.
1. Marcy, Y. et al. (2007) Dissecting biological ‘‘dark matter’’ with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth. Proc. Natl. Acad. Sci. USA 104, 11889. Link
2. Youssef, N.H. et al. (2015) Assessing the global phylum level diversity within the bacterial domain: A review. J. Adv. Res. 6, 269. Link
5. Wilke, A. et al. (2015) The MG-RAST metagenomics database and portal in 2015. Nucl. Acids Res. doi: 10.1093/nar/gkv1322 Link
6. Eloe-Fadrosh, E.A. et al. (2016) Global metagenomic survey reveals a new bacterial candidate phylum in geothermal springs. Nat. Commun. 7, Article number: 10476 doi:10.1038/ncomms10476 Link
Trademarks: REPLI-g® (QIAGEN Group); MiSeq® (Illumina, Inc.)