17- Metagenome

Question 1

define the term metagenomics

Answer 1

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

Question 2

define the term microbiome

Answer 2

is the community of microbiota (micro)organisms and their combined genetic material in a well-defined habitat

Question 3

define microbiota

Answer 3

the ecological community of commensal and pathogenic microorganisms – bacterial, archaea, protists, fungi and viruses – a combination of the organisms themselves

Question 4

describe human microbiome environments

Answer 4

human microbiome includes such things as the skin, nasal, oral vaginal microbiomes

within different microbiomes there’s a different distribution of different bacterial species unique to every individual - determined by genes, environment, medicines, food eaten…

Question 5

describe the relationship between microbiomes and disease with examples - C.difficile

Answer 5

changes in the microbiome are associated with certain diseases and can be used as diagnostic predictors

e.g. early-life microbiomes in babies have been linked to allergic conditions like bacteria

for C. difficile infections, the stool microbiome differs from heathy stool microbiome

Question 6

what are the 5 stages for 16srRNA targeted PCR amplification

Answer 6

sample collection
DNA extraction
PCR amplification
sequence matching
analysis

Question 7

describe specifically targeted PCR amplification as a technological approach to metagenomics

Answer 7

16s rRNA PCR amplification specifically targets the 16s region of the prokaryotic 30s small ribosomal subunit

targeted as it contains variable and conserved regions - variable regions contain a phylogenetic signal that differentiates bacterial species

workflow:
1. sample collection
separating the sample bacterial cells, different colours for different species

2. DNA extraction
   extracting DNA from the bacterial cells
3. PCR amplification
   - targeted, specific amplification of the 16s rRNA variable region
   - different PCR product produced for different bacterial species
4. sequence matching
   generating sequences for each of the 16s genes
5. analysis
   - comparing obtained sequences to 16s databases - e.g. Greengenes, Silva - to identify the species present in the original sample
   - can create relative abundance charts for the different bacterial species

Question 8

what two factors determine which variable region to choose?

Answer 8

two factors: phylogenetic signal, amplicon length

phylogenetic signal
- more phylogenetic signals within variable regions = easier to distinguish between different bacterial species, more ideal for PCR amplification

amplicon length
- shorter amplicon length = more overlap, easier resolving of single point errors, providing a better quality sample

we want a short amplicon length with many different phylogenetic signals

Question 9

what is a phylogenetic signal?

Answer 9

information contained in a genetic sequence reflecting its evolutionary history

Question 10

describe high-throughput short read sequencing platforms in 16S PCR amplification - examples?

Answer 10

e.g. Roche 454, Illumina HiSeq and MiSeq

different machines have different base pair read lengths, and give different outputs

short-read sequencing platforms can only sequence part of a gene - need to pick ideal variable regions

Question 11

what is the advantage and disadvantage of long-read high throughput sequencing?

Answer 11

pro: enables full length 16S sequencing

con: lots of noise, higher error rates

Question 12

factors that may affect 16S targeted amplification results? what controls can be used to mitigate these?

Answer 12

affecting factors:
- sequencing higher biomass samples to reduce contamination effects on sequencing results

• kiotome of labatory equipment = cross-over contamination can affect results

controls for mitigation:
- randomising samples
- noting batch numbers and reagents
- sequencing negative controls/contamination

Question 13

why is the kitome is an important consideration for metagenomics?

Answer 13

contamination of labatory equipment - it can contaminate the samples and affect the results of the experiment

kits will have a different profile of contamination from one another

Question 14

define 16S targeted PCR amplification

Answer 14

specific targeting of the 16s region of the prokaryotic 30s small ribosomal subunit for PCR amplification and sequence analysis, to differentiate between different bacterial species using phylogenetic signals

Question 15

define whole shotgun metagenomics

Answer 15

sequencing the genetic diversity of an entire microbial community, looking at all micro-organisms and their genetic info.

doesn’t rely on specific marker genes - e.g. 16S rRNA

Question 16

describe whole genome shotgun sequencing as a technological approach to metagenomics

Answer 16

sequencing the genetic diversity of an entire microbial community, looking at all micro-organisms and their genetic info.

doesn’t rely on specific marker genes - e.g. 16S rRNA

workflow:
1. sample collection
2. DNA extraction = genomic DNA is fragmented
3. whole genome sequencing = occurs through assembly or binning

assembly = small whole genome shotgun sequences become larger sequences through software
- used to build evolutionary trees, observe taxonomic diversity or build gene prediction profiles on identified genes and their biochemical pathways

binning = sequences compared to a database, different sequences put in different taxonomic groups

4. sequencing = with high-throughput tech.
5. analysis = identifying and characterising microbial genomes

Question 17

what are the 5 stages for whole shotgun metagenomics

Answer 17

sample collection
DNA extraction
whole genome sequencing
sequencing
analysis

Question 18

advantages of whole genome shotgun sequencing in metagenomics?

Answer 18

• comprehensive, unbiased view of the microbial discovery of new species and genes
• more in-depth genomic analysis

Question 19

disadvantages of whole genome shotgun sequencing in metagenomics

Answer 19

• host cells are often in excess in the sample
• no amplification to enrich bacterial DNA
• amount of contaminating DNA depends on the sample = more biomass means less contaminating reads

Question 20

how is a sample enriched without amplfiication?

Answer 20

two approaches - pre and post-extraction

pre-extraction = differential lysis of mammalian based on their biochemical properties
- using enzymes/ chemical to target and break down mammalian cell walls = leave bacteria cell walls intact
- potential bias towards gram-positive bacterial due to cell wall structure

post-extraction = the specific enzymatic breakdown of methylated nucleotides in mammalian DNA
- discriminated methylation patterns
- bias towards AT rich bacterial genome = discriminates methylation patterns less effectively

Question 21

describe metagenomic applications with environmental genomes - example?

Answer 21

in environmental microbiomes = e.g. Saragasso sequencing
- shotgun genome sequencing of environmental sequencing
- can identify new genes, species, biological pathways
- affects pharmaceutical drug development

Question 22

describe metagenomic applications with animal genomes - example?

Answer 22

in animal microbiomes = e.g. rumen cattle microbiome
- sequencing animal microbiome
- can sequence and reassemble whole genomes of various bacteria, understand animal microbial communities

Question 23

describe metagenomic applications with clinical/diagnostic microbiology

Answer 23

1. identifying hard to culture clinical samples directly = no need for isolation and culture
2. identifying antibiotic resistance
   - sequencing entire samples, identifying resistance genes
   - predicting sample/drug resistance profiles depending on the absence /presence of a resistance gene
   - e.g. beta-lactamase presence = penicillin resistance
3. comparing healthy and unhealthy microbes for diagnostics
4. public health
   - infection control and outbreak management = identifying the microbial component
   - surveying antimicrobial resistance = sequencing samples and analysing all resistance genes for a broader range of info.

Question 24

advantage of metagenomics in studying directly recovered genetic material

Answer 24

• unbiased view of taxonomic diversity in a sample
• not limited by ability to culture
• overall view of gene content in a sample