The Metagenome Flashcards
Define the following terms: Genomics, Transcriptomics, Proteomics, Metabolomics
- Genomics: Whole cell gene content
- Transcriptomics: Whole cell gene expression
- Proteomics: Whole cell protein content
- Metabolomics! Whole cell metabolite content
Why is it important to understand different organisms in an environment?
- Organisms do not live in isolation
- Most environment have a complex mixture of species
- Many organisms cannot be cultured
- Important to understand interactions within these complex mixtures
Define the term “Metagenomics”
The study of genetic material recovered directly from the environment samples (systems)
What is a micro biome?
A Microbial community living in a reasonably well defined habitat that has distinct physio chemical properties
What is Microbiota?
An ecological community of commensalism and pathogenic microorganisms (bacteria, archaea, protists, fungi, viruses)
What part of the Microbiomes are considered environmental?
- Deep sea microbiome
- Soil microbiome
- Hospital microbiome
- Subway microbiome
What part of the Microbiomes are considered human/living?
- Gut microbiome
- Skin microbiome
- Oral microbiome
- Vaginal microbiome
How does taxonomic diversity vary in the human microbiome?
Taxonomic diversity Varies by body site
What is the significance about the human microbiome?
- A microbiome is unique to each individual, even between twins
- Changes in the microbiome have been associated with multiple illnesses e.g cancer, depression
- Individuals with Gut microbiome can classify as lean or obese with >90% accuracy
- Early life gut microbiomes linked to development of allergic conditions
What are other significant factors about microbiomes?
- Stool microbiome during Clostridium difficile infection (CDI) are quite different from healthy stool
- CDI has greater effects on stool microbiome than host genetic factors
- Faecal microbiota transplant is able to cure CDI
- Restorwtion of the stool microbiome to that of healthy state is rapid following transplantation
What are the technological approaches of metagenomics?
- Using Targeted PCR amplification
- 16S rRNA, bacteria
- internal transcribed spacer
- Whole genome shotgun sequencing
What is 16S targeted PCR amplification?
16S ribosomal RNA is a component of 30S small subunit of prokaryotic ribosomes
Describe the steps involved in 16S targeted PCR amplification
Sample collection -> DNA extraction -> 16S PCR amplification -> Sequencing -> Analysis
How can the data from 16S targeted PCR amplification be analysed?
Using different softwares such as QIIME2, Mothur, DADA2
What are the high throughput sequencing machines?
- Roche 454
- Illumina HiSeq
- IonTorrent PGM
- Illumina MiSeq
- PacBio
- ONT MinION
Which ones are the short read sequencing?
- Roche 454
- Ion Torrent PGM
- Illumina MiSeq
- Illumina HiSeq
Which ones are the long read sequencing?
- PAC Bio
- ONT MinION
Which variable regions are chosen for the 16S targeted PCR amplification?
- Phylogenetic signal
- Amplicon length
What are the problems with these methods?
They’re very sensitive to
- Contamination
- Operator
- Reagents
They’re important for low biomass samples
Where are 16S rRNA genes found?
- 16S rRNA gene found in all bacteria
- Mitigate potential contamination
- Randomise Samples
- Note batch numbers of reagents
- Sequence negative controls
What is the problem with full length 16S sequencing?
- They produce High error rates
- Introduce noise
What is the advantage with using full length 16S sequencing?
- Increased throughput
- Reducing cost of sequencing technologies
- Reducing studies using 16S targeted PCR amplification
Describe the whole genome shotgun flow
- Host cell 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 sample: >90% human reads
What is one way you can enrich your sample without undergoing amplification?
Pre-Extraction
- Differential lysis of mammalian cells
- Enriches for intact microbial cells
- Potential bias towards gram positive bacteria
What is another way you can enrich your sample without undergoing amplification?
Post- Extraction
- Enzymatic degradation of methylated nucleotides target mammalian DNA
- Bias against AT rich bacterial genomes
Give 3 types of applications for Metagenomics
- Environmental
- Animal
- Clinical diagnostics
Briefly describe the applications of meta genomics in terms of environment
Environmental genome shotgun sequencing of the Saragossa sea
- 1.045 billion base pairs sequenced
- Elucidate the gene content, diversity and relative abundance of the organisms
Briefly describe the applications of meta genomics in terms of environment (PART 2)
- Estimated to derive from at least 1800 genomic species
- Identified 148 previously unknown bacterial phyllotypes
- Identified over 1.2 million previously unknown genes
Briefly describe the applications of meta genomics in terms of Animal Microbiomes
Rumen Microbiome
- First of four chambers in cow’s stomach
- Contains a mix of bacteria and other organisms which ferment complex carbohydrates to produce short chain fatty acids
- Generated 6.5 Tera bases short and long read sequences from 283 ruminant cattle
- Assembled 4941 genomes including 3 whole chromosomes assemblies of rumen bacteria
Briefly describe the applications of meta genomics in terms of Clinical meta genomics
Diagnostic Microbiology
- standard is to culture isolate and then identify using matrix assisted laser desorption/ionisation (MALDI)
- Many organisms can’t be cultured
- Can identify hard to culture organisms in patient samples
- Identify antibiotic resistance repertoires directly from clinical samples
Briefly describe the applications of meta genomics in terms of Clinical meta genomics (PART 2)
Diagnostic microbiology (cont.)
- Potential to develop diagnostics based on differences in micro biomes
Briefly describe the applications of meta genomics in terms of Clinical meta genomics (PART 3)
Public health
- Infection control and outbreak management
- Surveillance of anti microbial resistance in the food supply
