metagenome Flashcards
What is metagenomics?
- study of whole sequence in cell (overall view of gene content in sample)
- organisms do not live in isolation so genetic material is recovered from environmental or biological systems.
- unbiased view of taxanomic diversity in a sample
- not limited to ability to culture (some cant be cultured)
What is microbiota?
ecological community of commensal and pathogenic microorganisms including bacteria, fungi, viruses, etc
What is 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 activity.
What is human micro-biome?
- taxonomic diversity varies by body site
- unique to each individual (even twins)
- changes in micro biome can be associated with disease (IBS, cancer, depression)
- can classify humans as lean/obese (90%) accuracy
define the 4 ‘omics’
- genomic = whole cell gene count
- transcriptomics = whole cell gene expression
- proteomics = whole cell protein content.
- metabolomics = whole cell metabolite.
how can we use human microbes to identify disease?
- microbiome unique to each individual, even between twins.
- changes in the microbiome have been associated with multiple human illness, eg: irritable bowel syndrome, depression, cancer
- help classify individuals as lean or obese with >90% accuracy
- early life microbiomes linked to development of allergic conditions, eg. asthma
What is an example of disease which can be identified by looking at stool microbiome and how can you treat it?
- clostridium difficile infection (CDI)
- CDI has greater effect on stool than host genetic factors
cure: - faecal microbiota transplant is able from healthy patients
-restoration of the stool microbiome to that of healthy state is rapid following transplantation.
what are the steps to 16S targeted PCR amplification workflow?
- sample collection
- DNA extraction
- 16S PCR amplification
- sequencing
- analysing
What is the genetic make up of 16S ribosomal RNA?
- 16S ribosomal RNA is a component of 30S subunit of prokaryotic ribosome.
- about 1500 bases long
- separated into 9 variable regions
- has conserved regions too
What are the 5 common variable regions used for PCR?
V1- V2 V1- V3 V3 - V4 V3- V5 V4
Which phylogenetic signal tree separates species well?
=>V1- V2 separates species well
=> V4 doesn’t distinguish species well
How does amplicon length affect quality of data?
V1- V3 only partly overlaps
V1- V2 completely overlaps with V4
when variables overlap the quality of data is better.
What is the advantage of high throughput sequencing?
- the sequence length increases
- meaning we can genome the entire sequence
- much greater resolution.
Why must we use controls?
- method targeted PCR amplification is very sensitive to contamination so
- must control for contaminations coming from reagent used.
How do we mitigate potential contamination?
- randomise samples
- note batch numbers of reagent
- sequence negative control
compare the 2 technological approaches : targeted 16S PCR amplification and genome sotgun sequencing?
targeted 16S PCR :
=> asses taxanomic diversity in sample
=> biased, only bacteria
whole genome shotgun:
=> assess taxonomic diversity in sample
=> assess composite gene functions in sample
=> unbiased, all micro-organisms
What are problems with whole genome shotgun workflow?
=> host cells often in excess in the sample
=> no amplification step to enrich for bacterial DNA
=> sample dependent, typical yields of contaminating human reads :
feacal : <10%
saliva, nasal, skin samples : > 90% human reads
How do we enrich genome without amplification?
- pre-extraction
- differential lysis of mammalian
- enrich for intact microbial cells
- potential bias towards gram -positive - post -extraction
- enzymatic degradation of methylated nucleotides targets mammalian DNA
- Bias against AT rich bacterial genomes
What are 3 different meta-genomics applications?
- environmental
- animal
- clinical diagnostics
What are features of the environmental genome shotgun sequencing of the saragasso sea(2004)?
- 1.045 billion base pairs sequenced
- elucidate the gene content, diversity, and relative abundance of the organisms
- estimated to derive from 1800 genomic species
- identified 148 previously unknown bacterial phylotypes
- identified over 1.2 million previously unkown genes
What are features of animal microbiomes (Rumen microbiomes)?
- first of 4 chambers of cows stomach
- contains a mix of bacteria and other organisms which ferment complex carbohydrates to produce short chain fatty acids
- generated 6.5 terabases short and long read sequence ruminant cattle
- assembled 4,941 genomes including 3 whole chromosome assemblies rumen bacteria.
how can metagenomics be used clinically for diagnosis?
- gold standard is to culture isolate and then identify using matrix assisted laser desorption/ionisation (MALD)
- many organisms cannot be cultured
- can identify hard culture organisms in patient samples e.g neuroleptospirosis in 14 y/o critically ill boy with meningoencephalitis
- identify antibiotic resistance repertoires directly from clinical samples.
- potential to develop diagnostics based on differences in micro biomes.
How is clinical metagenomics used in public health?
- infection control and outbreak management
- surveillance of antimicrobial resistance in the food supply.
