long question (9) Flashcards
1
Q
describe the workflow involved in sequencing microbiota and why is it important
A
2
Q
the workflow involved in sequencing microbiota
A
- sample preparation & handling
- DNA extraction
- PCR & sequencing
- bioinformatics
- multivariate analysis
3
Q
sample preparation
A
- stabilising homogenised samples at 4C within 12 hours of retrieving
- biobanking (freeze + thawing 4 times or more) by storing sample in freezer at -80C & results in microbiota composition shifts
- propidium monoazide to remove dead cells, extracellular DNA
4
Q
DNA extraction
A
- using high-throughput DNA extraction kits e.g. RNeasy PowerMicrobiome kit
- cell walls need to be lysed in order to extract DNA
- some bacterial cell walls are difficult to lysis so that needs to be taken into account e.g. gram positive + Mycobacterium spp.
- run positive (mock communities) + negative controls
5
Q
PCR & sequencing
A
- using high-throughput PCR reaction kits e.g. Onar Bacteria Detection which detects >45 bacteria genera
- run positive (mock communities) + negative controls
- Illumina MiSeq can multiplex 384 samples
- 192 samples result in 50,000
6
Q
bioinformatics
A
- Illumina MiSeq produces millions of raw sequences pipelines
- organises those sequences back to corresponding sample
- remove poor sequences
- pipelines include QIIME (operational taxonomic units) + DADA2 on R platform (exact sequence variants)
7
Q
multivariate analysis
A
microbiota organisation
- diversity
- composition
- structure
8
Q
why is it important
A
- a lot of gut bacteria contribute to human diseases
- only 1% of microbes are culturable
- cultures can have different & respiratory conditions, can take 100s of agar plates
- metagenomics allows to investigate all micro-organisms in the gut
- gut-brain axis explores the relationship between the neurons & gut bacteria, microbiome can have connections to mental health
