The Metagenome Flashcards
As a recap, define:
- genomics
- transcriptomics
- proteomics
- metabolomics.
- Genomics: the whole cell gene content
- Transcriptomics: the whole cell gene expression
- Proteomics: the whole cell protein content
- Metabolomics: the whole cell metabolite content
Define metagenomics.
Metagenomicsis the study ofgeneticmaterial recovered directly fromenvironmentalor biological systems/compartments.
Define microbiota.
- the ecological community of commensal and pathogenic microorganisms
- this includes bacteria archaea, protists, fungi and viruses
Define microbiome.
The collective genomes of the micro-organisms in microbiota.
Describe the human microbiome.
The microbiome is 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).
The gut microbiome can classify individuals as lean or obese with >90% accuracy. Early-life gut microbiomes are linked to the development of allergic conditions (e.g. asthma).
Specifically, describe the human stool microbiome.
The stool microbiome during Clostridium Difficile Infection (CDI), quite different from healthy stool microbiome. CDI has a greater effect on the 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.
List the technological approaches to metagenomics.
- Targeted PCR amplification
(16S rRNA, bacteria - 18S rRNA, eukaryotes): this includes using a single marker that we know has some variation within a population between species. We use this as a ‘proxy’ to find out about the species and organisms in a sample. - whole genome shotgun sequencing
Describe the 16S targeted PCR amplification workflow.
- sample collection
- DA extraction
- 16S PCR amplification
- sequencing
- analysis
Describe the variable regions.
The variable regions are conserved within a phylum (genetic group), but they converge within a species.
These regions are used to separate species based on these sequences.
Which variable region do we choose (V1-V9)?
You would decide it based on the:
- phylogenetic signal: does the variable region contain enough information to separate the species/genera that you’re interested in separating from you sample?
- amplicon length: how big can the PCR product be?
Thus, it depends on the sample you’re analysing.
What could go wrong with 16S targeted PCR amplification?
The method is very sensitive to contamination from the:
- environment
- operator
- reagents
This is especially important for low biomass samples.
How would you mitigate the potential contamination of 16S targeted PCR amplification?
- randomise the samples: make sure all the conditions and the controls are random on the plate, and that it’s all done at the same time
- note the batch numbers of the reagents
- sequence to negative controls: this is to make sure that it’s from your kit
How does 16S targeted PCR amplification cope with hard reads?
The choice of variable region determines the resolution. It is less reliable below genus level.
Thus, 16S is hard with long reads.
There is new long read technology that enables full length 16S sequencing:
- PacBio
- Nanopore
However, the higher error rates of long read technologies introduce noise. The development for this technology is ongoing.
Describe whole genome shotgun sequencing.
Instead of doing a PCR, you’re using the whole DNA - so there is no bias.
By finding out which genes are present, we infer which metabolic pathway is present in the sample. As you compare with other samples, you’re able to look for enrichment, decreasing metabolic pathways between diff environments, niches, patients, etc.
List some characteristics of whole genome shotgun sequencing.
The host cells are often in excess in the sample. There is no amplification step to enrich for bacterial DNA.
It is sample dependent, and the typical yields of contaminating human reads are:
- faecal: <10% human reads
- saliva, nasal, skin samples: >90% human reads
