W4 - The Metagenome Flashcards
What is Metagenomics?
“Metagenomics is the study of genetic material recovered directly from environmental samples”
Metagenomics is the study of genetic material recovered directly from environmental or biological
systems/compartments
What is a 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” [1]
Microbiome adds all the other components of activity to those micro-organism in the microbiota?
What is Microbiota?
Ecological community of commensal and pathogenic microorganisms. Includes bacteria, archaea, protists, fungi and viruses
This is basically the combination of all the microorganisms in the sample.
What are examples of microbiomes? Environmental vs Human?
Environmental:
* Deep sea microbiome
* Soil microbiome
* Hospital microbiome
* Subway microbiome
Human:
* Gut microbiome
* Skin microbiome
* Oral microbiome
* Vaginal microbiome
In a 70kg human, about 1-3kg is made of bacteria. The types of bacteria in each site and quantity varies.
What does the Human Microbiome consist of?
- Microbiome unique to each individual, even between twins
- Changes in the microbiome have been associated with multiple human illnesses, e.g. Irritable Bowel Syndrome, depression, cancer. Each of these studies show correlation, not causation.
- Gut microbiome can classify individuals as lean or
obese with >90% accuracy - Early-life gut microbiomes linked to development of allergic conditions e.g. asthma
- Stool microbiome during Clostridium difficile infection
(CDI), quite different from healthy stoolmicrobiome. - CDI has greater effect on stool microbiome than host genetic factors
- Faecal microbiota transplant is able to cure CDI
- Restoration of the stool microbiome to that of healthy state is rapid following transplantation
How are Metagenomics approached?
Technological approaches
*Targeted PCR amplification
*16S rRNA is unique to, bacteria
*Internal transcribed spacer (ITS), 18S rRNA,
eukaryotes
*Whole genome shotgun sequencing
What is 16S targeted PCR amplification?
16S ribosomal RNA is component of 30S small subunit of prokaryotic ribosome
1)Sample collection
2)DNA extraction
3)16S PCR amplification
4)Sequencing
5)Analysis - the colours at this stage represents the different bacteria from the original sample.
We take these bacteria and compare them to the known 16S database: Greengenes, Saliva and RDP
Relative abundance of bacteria changes over time.
Which variable region should we choose in 16S targeted PCR amplification?
- Phylogenetic signal - Different variable regions are sequenced and their similarities are noted to create a diagram.
- Amplicon length - make sure there is as much overlap between sequences as possible to retain minimal errors.
What is the significance of “Read Length”?
All the samples that come out of the sequencing machines has different lengths.
What controls should be used?
16S rRNA gene found in all bacteria
* Method very sensitive to contamination
-Environment
-Operator
-Reagents
* Especially important for low biomass samples
* “Kitome” - reagent used can have contamination being carried over.
How do we mitigate potential contamination?
Mitigate potential contamination
* Randomise samples
* Note batch numbers of reagents
* Sequence negative controls - aka the contamination also gets sequenced so this can be taken into account.
How does the whole genome shotgun workflow work?
The whole genome is now sequenced as opposed to single gene. This can be done in different ways - the sample can be taken and created into an assembly. Binning is also another option where we take out sequences to compare to different data bases of taxonomic samples.
With the assembly, you can make taxonomic diversity groups and predict genes. With gene prediction, different chemical pathways present can be explored.
*Host cells often in excess in the sample
*No amplification step to enrich for bacterial DNA
*Sample dependent, typical yields of contaminating
human reads:
*Faecal: <10% human reads
*Saliva, nasal, skin samples: >90% human reads
How do you enrich it without amplification?
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
What are the applications of Environmental, Animal and Clinical diagnostics?
- Environmental
- Animal
- Clinical diagnostics
What are 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
