The metagenome Flashcards
What is genomics?
Whole cell gene content
What is transcriptomics?
Whole cell gene expression
What is proteomics?
Whole cell protein content
What is metabolomics?
Whole cell metabolite content
What is metagenomics?
It’s the study of genetic material recovered directly from environmental or biological systems
What is microbiota?
It is the ecological community of commensal and pathogenic microorganisms
Includes: bacteria, archare, protists and fungi
What is microbiome?
It is the collective genomes of the microorganisms in these communities
How is microbiomes to each individual and so what can we deduce?
Microbiome are unique to each individual, so more to do with environmental factors
What can gut microbiome in individuals classify?
Gut microbiome can classify individuals as lean or obese with >90% accuracy
What are early life gut microbiomes linked to?
Early life gut microbiomes linked to development of allergic conditions
What is the effect on stool microbiome during CDI?
Stool microbiome during clostridium infection is quite different from healthy stool microbiome
What does CDI have a greater effect on?
CDI has a greater effect on stool microbiome than host genetic factors
What can cure CDI?
Faecal microbiota transplant is able to cure CDI
What technological approaches to metagenomics can we use?
Targeted PCR amplification and we use 16S rRNA
What is 16s rRNA?
16s rRNA is a component of 30s small subunit of prokaryotic ribosome
What do we use to diversify between species?
We use the variable regions to diversify between species
What are the steps involved in targeted PCR amplification?
- We collect a mixed sample collection
- Then carry out DNA extraction of all bacteria
- Afterwards we do 16s PCR amplification
- We then put the PCR products in the sequencing machine and carry out analysis
What factors do we take into account when choosing the variable regions to choose in 16s PCR amplification?
- Phylogenetic signal
- Amplicon signal
What controls do we use in 16s PCR amplification?
We mitigate contaminations by:
- Randomise samples
- Note batch numbers of reagents
- Use sequence negative controls
What is whole genome shotgun technique?
Has the same steps as 16s PCR amplification but the whole genome is sequenced instead of only 16s rRNA
What do we to do the data we obtain from whole genome shotgun technique and how can we use this data?
The sequence from the sequencing machine is reassembled to make sense
- Can use the data to do a taxonomic diversity assessment
- Can also use the data for gene prediction
How do we enrich without amplification?(Pre extraction)
- Different lysis of mammalian cells
- Enrichs for intact microbial cells
- However potential bias towards gram positive bacteria
How do we enrich without amplification?(Post extraction)
- Enzymatic degradation of methylated nucleotides targets mammalian DNA
- Bias against AT rich bacterial genome
