Other Genomes In Our Body Flashcards
What is a meta genome
It is the genetic material in the environment
What is a microbiota
It’s the community of organisms
What is a micro biome
It is the collective genomes of microorganisms in an environment as well as their proteins and metabolites
What are the two metagenomic approaches
Targeted PCR amplification
This targets a single gene marker to identify variations between species e.g. 16s rRNA or 18s rRNA
Whole genome SHOTGUN sequencing
Why 16S rRNA PCR
16S = component of the 30S prokaryotic ribosome
Largely conserved with variable regions different between genus and species
The conserved regions allow primer design
Describe the use of the 16S variable regions
9 variable regions, combinations include:
V1-2, V1-3, V3-4, V4-5, V1-9
You choose the combination according to the regions that are different enough to differentiate species common in the tissue/environment of investigation
Smaller regions = better as NGS short reads are more likely to overlap (2*300bp)
Long read can be used but these have high error rates
What are the drawbacks of 16S targeted PCR amplification
It is sensitive to contamination as it can pick up bacteria on the environment, operator, reagent. This effect is larger when biomass is smaller
Biased to bacteria
It is not reliable below genus, and requires suitable choice of variable region
How can you mitigate contamination in 16S Targeted PCR amplification
Randomise samples - test controls and cases at same time
Note batch number of reagents - use same batch
Sequence negative controls - this effectively sequences any contamination on the reagents or tools
What is whole genome SHOTGUN sequencing
Sample > extract > sequence WITHOUT PCR > analyse
You can either reassemble genomes or simply bin reads into specifies to find out what the species are
What are the pros and cons of whole genome shotgun sequencing
There is no bias to a single organism
However host cells are often in excess and can overpower (depends on tissue e.g. faecal = <10%, while skin = >90% human cells)
How can you overcome whole genome shotgun limitations
Pre extraction = differential lysis of mammalian cells
This is biased to gram +ve bacteria as they have thick cell walls that can resist lysis
Post extraction = enzymatic degredation of methylated DNA (mammalian methylation pattern is different)
This is biased to AT rich bacterial genomes, with GC rich being weakest
Compare 16S targeted PCR amplification and whole genome shotgun sequencing
16S = taxonomic diversity BUT only bacteria and PCR introduces bias
WGSS = taxonomic diversity and gene function determined, and unbiased to species UNLESS enriched. Has high throughput for low cost
What is dysbiosis
This is when the microbiota changes which could be due to new eating habits or antibiotics
Why is the microbiome important
It is integral to host digestion
What is autochthonous and allochthonous bacteria
Autochthonous = resident Allochthonous = passenger
What are the benefits of a diverse microbiome
Increased energy and nutrient extraction via provision of unique enzymes e.g. to digest fibre
Alteration of appetitive signals
The competition of good V bad pathogen is protective to host
What can the microbiome tell you
It can identify if someone is lean or obese with 90% accuracy
Associated with IBD, depression, cancer etc. and allergic reactions e.g. asthma
Describe the role of the microbiome in IBS/IBD
Altering the microbiota through dietary changes, probiotics or antibiotics can be beneficial
The gut brain access can alter symptom perception in the brain
Whether IBS does not have a single cause so some patients benefit but it can be used as a diagnostic tool
IBD is inflammation in the gut - with environmental and genetic risk factors
How is the microbiome changed in colorectal cancer
Higher proportion of pseudomonas, heliobacter, acinetobacter
Less beneficial bacteria such as butyrate producing bacteria and bifidobacterium
