long question (9)

Question 1

describe the workflow involved in sequencing microbiota and why is it important

Question 2

the workflow involved in sequencing microbiota

Answer 2

• sample preparation & handling
• DNA extraction
• PCR & sequencing
• bioinformatics
• multivariate analysis

Question 3

sample preparation

Answer 3

• 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

Question 4

DNA extraction

Answer 4

• 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

Question 5

PCR & sequencing

Answer 5

• 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

Question 6

bioinformatics

Answer 6

• 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)

Question 7

multivariate analysis

Answer 7

microbiota organisation
- diversity
- composition
- structure

Question 8

why is it important

Answer 8

• 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