The Metagenome

Question 1

define these 4 words

Answer 1

genomics = whole cell gene content

transcriptomics = whole gene expression

proteomics = whole cell protein content

metabolomics = whole cell metabolite content

Question 2

define meta genomics

Answer 2

study of genetic material recovered directly from environmental samples

study of genetic material recovered directly from environmental or biological systems/compartments

Question 3

define microbiome

Answer 3

micro-organism involved encompasses their theatre of activity

Question 4

define microbiota

Answer 4

ecological community of commensal and pathogenic microorganisms, includes bacteria, archaea, protists, fungi and viruses

Question 5

describe environmental microbiomes

Answer 5

deep sea microbiome
soil microbiome
hospital microbiome
subway microbiome

Question 6

describe human microbiomes

Answer 6

gut
skin
oral
vaginal

Question 7

human microbiome p2

Answer 7

microbiome is unique to each individual
changes are associated with multiple human illnesses e.g. cancer
gut microbiome can classify individuals as lean or obese
early-life gut microbiomes linked to development of allergic conditions e.g. asthma

Question 8

human microbiome p3

Answer 8

stool microbiome during CDI = clostridium difficile infection

CDI has greater effect on stool microbiome than host genetic factors

faecal microbiota transplant able to cure CDI

restoration of the stool microbiome to that of healthy state is rapid following transplantation.

Question 9

describe 16s targeted PCR amplification

Answer 9

16s ribosomal rna is component of 30s small subunit of prokaryotic ribosome

Question 10

steps of 16S targeted per amplification workflow

Answer 10

sample collection
dna extraction
16s pcr amplification
sequencing 
analysis

Question 11

variable regions to choose for 16s targeted per amplification

Answer 11

phylogenetic signal

amplicon length

Question 12

info about 16s targeted per amplification

Answer 12

16s rRNA gene found in all bacteria

method v sensitive to contamination. = environment, operator, reagents

important for low biomass samples

Question 13

whole genome shotgun workflow

Answer 13

host cell in excess in 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%

Question 14

how to enrich without amplification

Answer 14

pre extraction =
differential lysis of mammalian cells
enrich for intact microbial cells
potential bias towards gram positive bacteria

post extraction =
enzymatic degradation of methylated nucleotides targets mammalian dna
bias against AT rich bacterial genomes

Question 15

metagenomic applications

Answer 15

environmental
animal
clinical diagnostics

Question 16

environmental microbiomes

Answer 16

Sargasso Sea

• 1.045 billions base pairs sequenced
• derive from 1800 genomic species
• identified 148 previously unknown bacterial phylotypes
• identified over 1.2 millionunkown genes

Question 17

animal microbiomes

Answer 17

rumen microbiome
- first 4 chambers of cow stomach
mix of bacteria and other organisms which ferment complex carbs to produce short chain fatty acids

generated 6.5 terabases short and long read

Question 18

diagnostic microbiology

Answer 18

gold standard used to culture isolate and identify using matrix assisted laser desorption

many organisms cannot be cultured

can identify hard to culture organisms in patient samples

identify antibiotic resistance repertoires directly from clinical samples

Question 19

clinical meta genomics

Answer 19

diagnostic microbiology = potential to develop diagnostics based on differences in microbiomes

public health =
infection control and outbreak management
surveillance of antimicrobial resistance in food supply