Metagenome Flashcards
What is genomics?
→ The whole cell content
What are early life gut microbiomes linked with?
→ Development of allergic conditions such as asthma
What is proteomics?
→ Whole cell protein content
What is metabolomics?
→ cell metabolite content
How do you purify DNA?
→ Culture the organism in isolation
Why in practise is DNA not able to be purified?
→ organisms do not live in isolation
→ they are a complex mixture of species
What is a microbiota and what does this include?
→ Ecological community of commensal and pathogenic microorganisms
→ bacteria, archae, prostists, fungi and viruses
What is a microbiome?
→ Collective genomes of the microorganisms in these communities
What diseases have changes in the microbiome been associated with?
→ Cancer
→ Depression
→IBS
What can gut microbiome classify?
→ individuals as being obese or lean
What are early life gut microbiomes linked with?
→ Development of allergic conditions such as asthma
What are the 4 human microbiomes?
→ Gut microbiome
→ Skin microbiome
→ Oral microbiome
→ Vaginal microbiome
What can cure a clostridium difficile infection and why?
→ Stool transplant
→ the microbiome in CDI is different to a healthy microbiome
Why is 16S targeted PCR used?
→ 16S is a ribosomal component of the 30S subunit in prokaryotes
→ all bacteria have the 16S subunit
Describe how 16S targeted PCR amplification works?
→ Sample (urine, blood, etc)
→ DNA is extracted from a mixed population of bacteria in the sample
→ 16S PCR amplification - the 16S gene amplifies
→ sequence the PCR
Why are there biases in 16S PCR?
→ Some bacteria purify better than others
→ some sequences amplify better than others
What is done with the sequences after 16S PCR?
→compared to a database of known 16S sequences
What bacteria changes during the first year of life and how?
→ Actinobacteria drops after the first years of life
What bacteria is present in babies when breastfeeding?
→ Bifidobacteria
What 2 things do you need to consider when doing PCR on a bacterial variable region?
→ If it contains enough information to separate the genus
→ amplicon length
Why is sequencing a smaller region better?
→ The sequencer can read it twice and any errors will cancel out because the two sequences are combined into one region
What are disadvantages to the 16S method?
→ It is sensitive to contamination
“Kitome” - reagents can have carry over contaminates
What is the 16S method used for?
→ Low biomass sample
How do you mitigate contamination?
→ Randomise the samples
→ Note batch numbers of reagents
→ Sequence negative controls
What are the disadvantages of long reads?
→ they have higher error rates and introduce noise
What is whole genome shotgun sequencing?
→ Sequencing all the genes not just the 16S
How does whole genome shotgun sequencing work?
→the entire genome is broken into small fragments of DNA
→short fragments are sequenced and assembled together by computer that find overlaps
→ you can assess the DNA taxonomically
→ overlay it on pathways
→ changes in metabolic pathways between species
What are you looking for in whole genome shotgun sequencing?
→ changes in metabolic pathways between species
What are the 3 issues with whole genome shotgun sequencing?
→ Host cells are in excess
→ no amplification step to enrich for bacterial DNA
→ sample dependent
How do you enrich without amplification (pre extraction)?
→ Differential lysis of mammalian cells - bacteria have different cell walls
→ put reagents to lyse the mammalian cells but not bacteria
Why can there be a bias with lysing mammalian cells?
→ Bias towards gram +ve bacteria which have a thicker cell wall
How do you enrich without amplificaiton (post extraction)?
→ Enzymatic degradation of methylated nucleotides which target mammalian DNA
→ bias against AT rich bacterial genomes
How is metagenomics used in diagnostic microbiology?
→culture isolate and then identify using matrix assisted laser desorption/ionization (MALDI)
→Can identify hard to culture organisms in patient samples
How can metagenomics be used in public health?
→Infection control and outbreak management
→Surveillance of antimicrobial resistance in the food supply
How do you decide which variable to choose?
Phylogenetic signal- (shorter sample will correct mistakes as read twice/overlap).
Amplicon length- changes with intro of new sequencing platforms
What are the two ways of assessing shotgun sequences?
- Assembly
2. Binning
What is binning?
process of grouping reads orcontigsand assigning them to individualgenome by comparing to databases
