Lecure 20 Flashcards
Proire to genetic sequencing technologies, _____ discovery took a long time
Pathogen
E.g HIv was first discovered in 1981 but we know it existed before that
How did we know HIV existed before it was discovered
- look at the root of the tree root
Can calibrate the time of divergence by looking at the time the samples were taken and estimating the mutation rate - the branch of the phylogenetic trees representing divergence over time - draw linear regression from clusters and where it crosses the X axis is when HIV had emerged into the population
Pathogen discovery was revolutionaised when gene sequencing was introduced
- used to have to grow the virus in the lab to see what was causing the disease (but most viruses are not culturable - dont know the right conditions)
- can use consensusPCR to test for presence of a particular virus, consesus PCR is when you can take a conserved region of that viruses genome and test for its presence in an assay (but can’t discover new pathogens, only when u know what ur looking for)
- metagenomics allows you to look at the whole tree
This is kinda yap
Why use genomic sequencing ?
- microbes can be difficult to culture
- a (sort of) unbiased way to study ALL microbes in a sample, not just those u can culture (our ability to recognise what is in there is hindered)
- allows us to understand the structure and function of microbial communities
- it is realivelt cheap
What is the infectome
What we call the way of studying an entire microbial community
What is the contents of the infectome
Bacteria + archaea + fungi + viruses
What kind of sequencing can u use to target sequencing the bacteria+ archea part of the infectome
16s rRNA sequencing
What kind of sequencing can u use to target sequencing the fungi part of the infectome
18S rRNA sequencing
What kind of sequencing can u use to target sequencing the whole infectome
Metagonomic / metatrasncriptomic sequencing
What is the difference between metagenomics and metatranscriptiomics?
Metagenomcis refers to DNA sequencing where as metatransctiptiomics refers to RNA sequencing
Metagenoics will tell u anything that had a DNA genome that is there
But a lot of DNA don’t transcribe DNA- only RNA so if we use metatransciptomics it will tell us anything there that is transcribing - including RNA
What does infectome analysis using metagenomics/ metatranscriptomics tell us?
Who’s there
- toxonomic classification
- population analysis
What can they do?
- functional analysis
What sequencing gives us an understanding of community structure - if its bacteria
16S rRNA sequencing
What gives us a better understanding of the metabolic potential of a community?
Metagenomics
What is metatranscriptomics?
- total RNA sequencing (RNASeq)
- it gives us the gene expression of microbes within natural environments
What does metatranscriptomics allow us to do?
Allows us to obtain whole gene expression profiling of complex microbial communities
What is the difference between metagenomics and metatranscriptomics?
Metagenomics = genetic content, identifying microbes present within a community
Metatranscriptomics = diversity of the active genes within such community, quantify their expression levels and monitor how these levels change in different conditions
What is the advantage of metatranscriptomics over metagenomics
It can provide information about the differences in the active functions of microbial communities which appear to be the same in terms of microbe composition
(This is important as u may have a diseases and heathy population and they may have the same community composition however if you do metatransciptomics diseased may have a much higher gene expression of a particular thing)
Metatransciptomics can be used to study the…
Virosphere (but also all the parasites, bacteria, fungi = infectome) as well as the host gene expression
Things to keep in mind - caveats
There is no standard way to produce metatranscripomical date
- thus if we want to compare studies of different samples we have to understand the caveats:
E.g
- sewuwncing platforms may vary
- coverage/depth may vary
- sample collection and preservation (RNA is very degradable)
- RNA/DNA extraction methods
- enrichment or depletion steps
- one sample is representive of a single time point
Sequence analysis : assembling the metagenomics - De novo assembly…
putting short sequence reads together to reconstruct longer sequences (contigs)
What are contigs?
Continuous sequences from shorter, overlapping reads
Once you’ve done de novo assembly, what do you do next?
Annotating: once assembled, ideniftying where the contigs came form using sequence homology
OR
Map sequence reads to a reference sequence (if you know what your looking for)
De novo assembly vs mapping
