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What we doMicrobial Single-Cell Genomics

Microbial single-cell genomics represents an innovative approach to study microbial diversity and symbiosis. It allows us to perform a targeted recovery of nearly complete genomes of microbes with specific features of our interest, or detect relationships between microbes found in close proximity in an environmental sample (one microbe inside of the other, or attached to each other).

Microbial single-cell genomics largely differs from the single-cell genomics widely used for gene expression analysis in cancer cells. Expressed genes in eukaryotic organisms can be amplified through oligo(dT) primers, however, this is impossible in the case of bacteria, which lack the poly(A) tails in their transcripts.

In the typical microbial single-cell genomics workflow, fluorescence activated cell sorting (FACS) is used to collect bacterial cells of interest, based on their cell size, internal granularity or fluorescence, which is analysed by the FACS instrument at the speed of several thousands of cells per second. The cells are sorted into 96 or 384 well plates and DNA is released from the cells by alkaline lysis. Afterwards, a mixture of random hexamers and phi29 polymerase is applied to the single cells in an isothermal reaction of 4-12 hours to enrich the DNA by whole genome amplification (WGA). The femtograms of DNA from one cell are amplified up to the quantities required by the standard sequencing library preparation kits. Content of each single-cell is sequenced separately resulting in so called single amplified genomes (SAGs). The sequencing output is analysed for the genome completeness and contamination. Afterwards, we can analyse the metabolic features of the collected microbes, or focus on their interactions with other microbes which were, such as infecting bacteriophages or attached symbionts. Many new genomes, new features and new microbial interactions would not be discovered, if single-cell genomics is not used.

Our group masters a wide variety of genomic techniques. For example, we often combine the single-cell genomics workflow with metagenomics, which is a shotgun sequencing approach providing us with insights into the whole microbial community composition and genomic features of metagenome assembled genomes (MAGs) obtained by contig binning. If the samples contain a lot of host cells, such as tissues from animals or humans, we assess the microbial composition by 16S rDNA amplicon sequencing. In addition, in order to verify our sequence-based observations we often use traditional cultivation techniques.

We have collaborators around the world, e.g.:

  • DOE Joint Genome Institute, Lawrence Berkeley National Laboratory, USA
  • Department of Chemistry and Biochemistry, University of California San Diego, USA
  • Bigelow Laboratory for Ocean Sciences, USA
  • Beth Israel Deaconess Medical Center, Harvard Medical School, USA
  • Australian Centre for Ecogenomics, University of Queensland, Australia
  • Institute of Molecular Biomedicine, Commenius University, Slovakia

About the group leader

Mária DžunkováMária Džunková got her first experience with the high throughput DNA sequencing at the Czech Academy of Sciences at the time when she  studied at the University of Life Sciences in Prague (2005-2010). Then she moved to Spain for her PhD studies at the University of Valencia (2010-2016) which included a research stay at the Harvard University (2014). After defending her PhD thesis entitled ”Metagenomics of the Human Gut Microbiome Directed by the Flow Cytometry” she got a postdoc position at the Australian Centre for Ecogenomics at the University of Queensland (2016-2019), where she developed “single-cell viral tagging” method for exploring the phage host range without the need for microbial culturing and applied it to the human gut microbiome. She was invited to a second postdoc (2016-2021) at the DOE Joint Genome Institute (Lawrence Berkeley National Laboratory) to continue with her microbial single-cell genomics techniques. In California she studied the relationships between the phages and bacteria in environmental samples and explored new targeted single-cell genomics techniques for discovering symbiotic microbes of the marine soft-bodied animals capable of producing bioactive molecules. She joined the Institute for Integrative Systems Biology (I2SysBio) in December 2021 to set up her Microbial Single-Cell Genomics research group.