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Microbiomes > L2: The Study of Microbiomes: omic and other techniques > Flashcards

L2: The Study of Microbiomes: omic and other techniques Flashcards

1
Q

what are the levels of microbiome study?

A
  1. who are they
  2. what do they do
  3. how do they do
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2
Q

levels of microbiome study: who are they - how is this answered

A

through taxonomic profiling

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3
Q

levels of microbiome study: what do they do- how is this answered

A

through microbiome functional profiling

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4
Q

levels of microbiome study: how do they- how is this answered

A

through mechanism study

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5
Q

how can we study microbiomes

A
  1. bacterial isolation
  2. 16S rRNA sequencing
  3. metagenomic sequencing
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6
Q

studying microbiomes - bacterial isolation

A
  • isolate → whole-genome sequencing
  • helps understand what microbes are there
  • only helps with taxonomic profiling (level one of microbiome study)
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7
Q

studying microbiomes: bacterial isolation - explain the process

A
  • extract DNA
  • amplify gene marker
  • sequence DNA
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8
Q

studying microbiomes: bacterial isolation - disadvantage

A
  • dark matter cannot be isolated
  • for these microorganisms, need to use metagenomic or 16S rRNA gene sequencing
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9
Q

studying microbiomes: bacterial isolation disadvantage - dark matter

A
  • used to describe the majority of microbial organisms that microbiologists cannot culture in a lab
  • makes up most microbes (only 1% can be cultured and isolated)
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10
Q

studying microbiomes: 16S rRNA sequencing - general process

A

microbiome → phylogenetic marker gene sequencing (amplicon) → taxonomic profile → predicted functional profile

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11
Q

studying microbiomes: 16S rRNA sequencing - why use 16S rRNA

A
  • it is part of prokaryotic small ribosomal subunit
  • has 9 conserved and hypervariable regions
  • low mutation and horizontal gene transfer rates
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12
Q

16S rRNA sequencing: why use 16S rRNA - what is the conserved regions used for

A

used to design primers which amplify genes

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13
Q

16S rRNA sequencing: 16S rRNA conserved regions - what must you do when designing primers and why

A
  • choose primers that overlap the paired hypervariable regions
  • full length variables are not used unless you only use one organism bc otherwise it will increase sequencing errors
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14
Q

16S rRNA sequencing: why use 16S rRNA - explain the hypervariable regions

A

allows us to see which genes are different

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15
Q

16S rRNA sequencing: why use 16S rRNA - what do conserved and hypervariable regions look like on a sequencing graph

A
  • conserved: low lines
  • hypervariable: high lines
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16
Q

studying microbiomes: 16S rRNA sequencing - explain the overall 16S rRNA analysis

A

environmental samples → bacteria + PCR amplification → 16S rRNA sequencing → sequencing comparison → community composition + phylogeny → diversity + statistical analysis

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17
Q

16S rRNA sequencing: overall 16S rRNA analysis - which parts are considered quantitative analysis on taxonomic groups

A

community composition + phylogeny → diversity + statistical analysis

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18
Q

16S rRNA sequencing:16S rRNA analysis - what are ASVs

A
  • Amplicon Sequence Variants
  • it is all the different DNA sequences within the sample
  • put the bacteria in a phylogeny and every node is a ASVs
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19
Q

16S rRNA sequencing:16S rRNA analysis - what are OTUs

A
  • Operational Taxonomic Unit
  • clustering sequences in a 97% similarity threshold
  • the old way
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20
Q

16S rRNA sequencing:16S rRNA analysis - what is a problem associated with OTUs

A
  • some groups don’t represent the threshold
  • certain genotypes that are different would represent the threshold
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21
Q

16S rRNA sequencing - 16S rRNA analysis limitations

A
  • restricted to prokaryotes since you will need to change the primers for eukaryotes
  • limited resolution and low sensitivity in some bacterial groups
  • PCR errors
  • results depend on the variable sequence considered
22
Q

16S rRNA sequencing: 16S rRNA analysis limitations - why may you have limited resolution and low sensitivity

A
  • some bacteria can only be sequenced to the genome level
  • cannot distinguish on the species level
23
Q

16S rRNA sequencing: 16S rRNA analysis limitations - why are the results dependent on the variable sequence

A

mutation rates across variable regions are different for each organism

24
Q

16S rRNA sequencing - is amplicon sequencing the best for showing true diversity

A
  • no
  • when comparing taxonomic results with metagenomic results, the metagenome always shows more diversity
  • this is bc not all groups can be shown through PCR
25
Q

16S rRNA sequencing - most common plots derived from 16S rRNA

A
  1. bar plots
  2. orgination (beta-diversity)
  3. boxplots (alpha-diversity)
26
Q

16S rRNA sequencing: most common plots - bar plots

A
  • shows dominant sample
  • color = bacterial groups
  • bars = different organisms
27
Q

16S rRNA sequencing: most common plots - bar plots disadvantage

A

when plotting a lot of organisms, it can look messy + you cannot see a consensus main groups

28
Q

16S rRNA sequencing: most common plots - orgination (beta-diversity)

A
  • compares different samples based on microbiomes
  • every dot = different sample/individual
  • the closer the dots are, the more similar the microbiomes are
29
Q

16S rRNA sequencing: orgination (beta-diversity) - explain the axis

A
  • amount of variation explained
  • percent shows the similarity
  • subtracting percent from 100, get the percent differences
30
Q

16S rRNA sequencing: most common plots - boxplot (alpha-diversity)

A

counting how many species (diversity) seen in each sample

31
Q

studying microbiomes - metagenomics

A
  • microbiome → shotgun sequencing → taxonomic + functional profile
  • sequences entire DNA samples without PCR
32
Q

studying microbiomes: metagenomics - explain the actual process

A

DNA extraction → fragmentation → DNA sequencing → read classification → taxonomic profiling OR virulence factor analysis OR functional profiling

33
Q

metagenomics: the actual process - why is it fragmentated

A

DNA sampling works better with fragments and not whole DNA samples

34
Q

metagenomics: explain the actual process - how is DNA sequencing done

A

via curated genomes from database

35
Q

metagenomics: the actual process - what is considered quantitative metagenomics

A
  • taxonomic profiling
  • virulence factor analysis
  • functional profiling via annotating the config to its function
36
Q

metagenomics: quantitative metagenomics - why is the ‘read classification’ step necessary before using quantitative metagenomics

A
  • in order to use quantitative metagenomics, you need to assemble DNA into larger configurations (config) via that step
  • so you have to generate a config
37
Q

metagenomics - explain metagenomic binning

A

its a process used to classify DNA sequences obtained from metagenomic sequencing into discrete groups (bins) based on similarity

38
Q

metagenomic binning - explain the process

A

DNA extraction → fragmentation → DNA sequencing → assembly → gene finding + annotation → phylogenetic binning (separates genomes) → metabolic reconstruction

39
Q

limitation of metagenomics

A
  • takes a lot of time, not feasible for large-scale studies + its expensive
  • assembling reads is impossible for medium or high diversity communities
  • may show host DNA in symbiotic systems (i.e., contamination)
40
Q

limitation of metagenomics - what solution is there for symbiotic systems

A
  • functional inference from 16S data
  • not an ideal solution however
41
Q

what are microbial “omics”

A
  • carious disciplines in biology
  • ex: metagenomics, metatranscriptomics, metaproteomic, metabolomics
42
Q

microbial “omics” - metagenomics

A
  • DNA level
  • asks:
    1. which microbes are present
    2. what is their relative abundance
    3. what microbial genes are present
43
Q

microbial “omics” - metatranscriptomics

A
  • mRNA level
  • asks:
    1. which microbial mRNA are present
    2. what are their potential abundance
    3. what is their relative abundance
    4. what microbes produced them
44
Q

microbial “omics” - metaproteomics

A
  • protein level
  • asks:
    1. which microbial proteins are present
    2. how much of the proteins are present
    3. what are their known functions
    4. which microbes express them
45
Q

microbial “omics” - metabolomics

A
  • metabolites level
  • asks:
    1. which microbial metabolites are present
    2. how much of the metabolites are present
    3. what are their known functions
    4/ which microbes produce them
46
Q

explain the Fluorescence in situ hybridization (FISH) protocol

A
  1. permeabilization/fixation
  2. hybridization
  3. washing
  4. visualization
47
Q

Fluorescence in situ hybridization (FISH) protocol - permeabilization/fixation

A

lets molecules have access to cells without ruining the cell

48
Q

Fluorescence in situ hybridization (FISH) protocol - hybridization

A

target a particular molecule/bacteria

49
Q

Fluorescence in situ hybridization (FISH) protocol - visualization

A

see green molecules through fluorescent microscope

50
Q

explain the nanoSIMS protocol

A
  • incubate samples and provide a nutrient and label it with an isotope
  • can now track it
  • more coloration = more nutrient present

Microbiomes (12 decks)

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