Metagenome Flashcards
Genomics
Whole cell gene content
Transcriptomics
Whole-cell gene expression
Proteomics
Whole cell protein content
Metabolomics
Whole-cell metabolite content
Metagenomics
is the study of genetic material recovered directly from environmental or biological systems/compartments
Microbiome
“a characteristic microbial community occupying a reasonably well-defined habitat which has distinct physio-chemical properties. The term thus not only refers to the microorganisms involved but also encompasses their theatre of activity”
Microbiota
• ecological community of commensal and pathogenic microorganisms. Includes bacteria, archaea, protists, fungi and viruses
Human microbiome
Gut Skin Oral Vaginal unique to each individual
Prokaryotic ribosome
50S subunit - 5s and 23S
30S subunit - 16s
Eukaryotic ribosome
60s subunit - 5s and 28s
40s subunit - 18s
Workflow
Sample collection, DNA extraction, 16S PCR amplification, sequencing, analysis
WGS shotgun
The assembly then either taxonomic diversity,
or
Gene prediction, pathways
Pre extraction
- Differential lysis of mammalian cells
- Enrichs for intact microbial cells
- Potential bias towards gram-positive bacteria
Post extraction
- Enzymatic degradation of methylated nucleotides targets mammalian DNA
- Bias against AT rich bacterial genomes
How to enrich without amplification?
Pre-extraction and Post -extraction
Metagenomics applications
- Environmental
- Animal
- Clinical diagnostics
Environmental microbiomes
- 1.045 billion base pairs sequenced
- elucidate the gene content, diversity, and relative abundance of the organisms
- estimated to derive from at least 1800 genomic species
- identified 148 previously unknown bacterial phylotypes
- identified over 1.2 million previously unknown genes
Animal microbiome
- First of four chambers of cow’s stomach
- Contains a mix of bacteria and other organisms which ferment complex carbohydrates to produce short chain fatty acids
- Generated 6.5 terabases short and long read sequence from 283 ruminant cattle
- Assembled 4,941 genomes including 3 whole-chromosome assemblies of rumen bacteria
Diagnostic microbiology
- Gold standard is to culture isolate and then identify using matrix assisted laser desorption/ionization (MALDI)
- Many organisms cannot be cultured
- Can identify hard to culture organisms in patient samples e.g. neuroleptospirosis in a 14-year-old critically ill boy with meningoencephalitis
- Identify antibiotic resistance repertoires directly from clinical samples
- Potential to develop diagnostics based on differences in microbiomes
Public
- Infection control and outbreak management
* Surveillance of antimicrobial resistance in the food supply
Technological approaches
Targeted 16S PCR amplification • Assess taxanomic diversity in sample • Biased, only bacteria Whole genome shotgun sequencing • Assess taxanomic diversity in sample • Assess composite gene functions in sample • Unbiased, all micro-organisms
