L7-9: Metagenomics, Gene regulation, Evolution genomics, Glycosylation Flashcards
What is metagenomics?
The study of genetic material recovered
directly from environmental samples such as gut, soil and
water.
Microbiome
The collection of genomes from all the microorganisms in
the environment
Microbiota
The microorganisms found in an environment
Functional genomics method
- Metagenomic DNA is isolated from microbial communities.
- DNA is then fragmented, cloned into vectors and amplified in a bacterial host.
- Functional screening of the metagenomic library then occurs
Sequence based metagenomics methods
16S rRNA amplicon sequencing
Whole genome shotgun sequencing
Why 16S rRNA gene is used for phylogenetic studies?
- It is present in almost all bacteria
- The function of the 16S rRNA gene over time has not changed, suggesting that random sequence changes are a more accurate measure of time (evolution);
- The 16S rRNA gene (1,500 bp) is large enough for informatics purposes
16S rRNA AMPLICON SEQUENCING - METHOD
- Sample collection
- DNA extraction
- PCR: Full length 16s rRNA or subset of the nine specific regions: V1-V3, V3-V5, V4-V5, V6-V9 etc.
- Sequencing: sequencing technology e.g. Illumina Miseq
- Bioinformatics:
- Quality control: trimming low quality reads
- Assigning OTUs (Operational Taxonomic Unit)
Advantages and disadvantages of 16S rRNA sequencing for community taxonomic
composition studies
Advantages: - Efficient - Cost effective - Taxonomic composition - Relative abundance Disadvantage: - Low taxonomic resolution - Chimeric amplicons.
What is PCR chimera?
An artifactual PCR product/amplicon generated erroneously from more than one DNA template
Detect and filter them out before any types of microbiome analyses.
Whole genome shotgun sequencing - method
- Sample collection
- DNA extraction
- Construction of a metagenomic library: e.g using Nextera DNA Flex:
- Bead-linked transposomes mediate the fragmentation of gDNA and the addition of sequencing primers
- Reduced cycle PCR amplifies sequencing-ready DNA fragments and adds indexes and adaptors - Sequencing: High throughput Illumina platform e.g Illumina HiSeq
- Assemble to reconstruct the genomes of the organisms in the sample or assembly-free for taxonomy characterisation of metagenomes:
- classified into taxonomic group through comparison to a reference data
- clustered into groups based on shared characteristics - Functional profiling: mapping the gene sequencings against databases of functional genes. Reveals the presence or absence of biological pathways, the evolution
and the survival strategies of the community microorganisms. Pathways analysis by using annotated database such as KEGG, PFAM, UNIREF etc.
Pros and cons of WGS sequencing
Advantage • Higher resolution • Taxonomic composition • Relative abundance • Functional potential Disadvantage • Expensive • High sequencing depth required
Alpha-diversity
Within-community diversity:
i.e., the number of species and their proportion within one sampling site.
Beta diversity
Between-community diversity:
-The higher beta diversity, the more dissimilar the two communities
Application of metagenomics
Biofuel: fuels derived from biomass conversion
- Microbes involve in the production of a variety of sources such as ethanol, methane,
hydrogen
- Mining complex microbial communities for novel biocatalytic enzymes (Hess et al 2011)
- Metagenomic discovery of biomass-degrading genes and genomes from cow rumen
Gut Microbe Characterisation
- Play an important role in protecting the host against pathogenic microbes, modulating immunity, regulating metabolic processes etc.
- The change in the human microbiome can be correlated with human health
Human IgG is a protective antibody that contains a conserved ______ site in its ____ region
N-glycosylation; Fc
