L2: The Study of Microbiomes: omic and other techniques

Question 1

what are the levels of microbiome study?

Answer 1

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

Question 2

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

Answer 2

through taxonomic profiling

Question 3

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

Answer 3

through microbiome functional profiling

Question 4

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

Answer 4

through mechanism study

Question 5

how can we study microbiomes

Answer 5

1. bacterial isolation
2. 16S rRNA sequencing
3. metagenomic sequencing

Question 6

studying microbiomes - bacterial isolation

Answer 6

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

Question 7

studying microbiomes: bacterial isolation - explain the process

Answer 7

• extract DNA
• amplify gene marker
• sequence DNA

Question 8

studying microbiomes: bacterial isolation - disadvantage

Answer 8

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

Question 9

studying microbiomes: bacterial isolation disadvantage - dark matter

Answer 9

• 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)

Question 10

studying microbiomes: 16S rRNA sequencing - general process

Answer 10

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

Question 11

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

Answer 11

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

Question 12

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

Answer 12

used to design primers which amplify genes

Question 13

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

Answer 13

• 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

Question 14

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

Answer 14

allows us to see which genes are different

Question 15

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

Answer 15

• conserved: low lines
• hypervariable: high lines

Question 16

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

Answer 16

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

Question 17

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

Answer 17

community composition + phylogeny → diversity + statistical analysis

Question 18

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

Answer 18

• 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

Question 19

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

Answer 19

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

Question 20

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

Answer 20

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

Question 21

16S rRNA sequencing - 16S rRNA analysis limitations

Answer 21

• 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

Question 22

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

Answer 22

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

Question 23

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

Answer 23

mutation rates across variable regions are different for each organism

Question 24

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

Answer 24

• 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

Question 25

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

Answer 25

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

Question 26

16S rRNA sequencing: most common plots - bar plots

Answer 26

- shows dominant sample - color = bacterial groups - bars = different organisms

Question 27

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

Answer 27

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

Question 28

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

Answer 28

- compares different samples based on microbiomes - every dot = different sample/individual - the closer the dots are, the more similar the microbiomes are

Question 29

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

Answer 29

- amount of variation explained - percent shows the similarity - subtracting percent from 100, get the percent differences

Question 30

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

Answer 30

counting how many species (diversity) seen in each sample

Question 31

studying microbiomes - metagenomics

Answer 31

- microbiome → shotgun sequencing → taxonomic + functional profile - sequences entire DNA samples without PCR

Question 32

studying microbiomes: metagenomics - explain the actual process

Answer 32

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

Question 33

metagenomics: the actual process - why is it fragmentated

Answer 33

DNA sampling works better with fragments and not whole DNA samples

Question 34

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

Answer 34

via curated genomes from database

Question 35

metagenomics: the actual process - what is considered quantitative metagenomics

Answer 35

- taxonomic profiling - virulence factor analysis - functional profiling via annotating the config to its function

Question 36

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

Answer 36

- 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

Question 37

metagenomics - explain metagenomic binning

Answer 37

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

Question 38

metagenomic binning - explain the process

Answer 38

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

Question 39

limitation of metagenomics

Answer 39

- 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)

Question 40

limitation of metagenomics - what solution is there for symbiotic systems

Answer 40

- functional inference from 16S data - not an ideal solution however

Question 41

what are microbial "omics"

Answer 41

- carious disciplines in biology - ex: metagenomics, metatranscriptomics, metaproteomic, metabolomics

Question 42

microbial "omics" - metagenomics

Answer 42

- DNA level - asks: 1. which microbes are present 2. what is their relative abundance 3. what microbial genes are present

Question 43

microbial "omics" - metatranscriptomics

Answer 43

- 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

Question 44

microbial "omics" - metaproteomics

Answer 44

- 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

Question 45

microbial "omics" - metabolomics

Answer 45

- 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

Question 46

explain the Fluorescence in situ hybridization (FISH) protocol

Answer 46

1. permeabilization/fixation 2. hybridization 3. washing 4. visualization

Question 47

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

Answer 47

lets molecules have access to cells without ruining the cell

Question 48

Fluorescence in situ hybridization (FISH) protocol - hybridization

Answer 48

target a particular molecule/bacteria

Question 49

Fluorescence in situ hybridization (FISH) protocol - visualization

Answer 49

see green molecules through fluorescent microscope

Question 50

explain the nanoSIMS protocol

Answer 50

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