Meta-Omics

Question 1

What is amplicon sequencing (16S gene) for?
What is shotgun sequencing for?

Answer 1

Amplicon - Metataxonomics
Shotgun - Metagenomics

Question 2

What was done before high-throughput sequencing to do microbiome research?

Answer 2

Do 16S PCR, run DNA product across gradients and it would migrate out to form bands
Separation of genes with mutations
- Diversity

Question 3

What did scientists do when they sailed around the world?

Answer 3

Sampled the oceans for microbial DNA for shotgun sequencing
Drastically changed the number of proteins in genomic databanks

Question 4

How did marine microbiome differ to soil microbiome research?

Answer 4

Marine microbiology – Frontier of microbial ecology and technology; Using metagenomics approach

Soil microbiology – Lagged behind due to the complexity/diversity of soil microbiome and contaminating substances

Question 5

What advancements allowed for the rise of metagenomics?

Answer 5

Prices of sequencing dropped massively
Computational power, bioinformatics, sequencing technology all improving

Question 6

Amplicon vs Metagenomic Sequencing (hint - single vs all)

Answer 6

Amplicon involves sequencing several copies of reads from 1 target gene in a mix of many fragments
- Use primers to do PCR of gene of interest

Metagenomics is to do with sequencing short sequence from all the DNA in an environmental sample

Question 7

What is meant by “no genomic context” as a disadvantage for amplicon sequencing?

Answer 7

Certain genes move around on plasmids and mobile genetic elements; Not part of chromosomes

If you sequence these out of the environment, you have no genomic context for where these things are

These genes move around via horizontal gene transfer; Sequence phylogenies won’t match organisms they’re coming out of

Question 8

Disadvantages of metagenomics? (2 main ones)

Answer 8

More computational power
Mis-annotation of functional genes; Assigns a gene a function which is incorrect

Question 9

Amplicons sequencing uses degenerate primers. What are these?

Answer 9

Degenerate primers target regions of high conservation like active sites or specific folding regions; Regions encoding function

Within this region there is a degree of variability; Degenerate primers are mixtures of similar primer sequences that take this variability in specific regions into account

Question 10

What are the 2 problems with amplicon sequencing? (hint - primers)

Answer 10

Primer bias; Primers may only be biasing towards 50,000 and not picking up the other 50,000

Need to know what you’re looking for so you can develop specific primers

Question 11

What is the top-down approach for metagenomics?

Answer 11

Not looking at specific function
Sequence everything and see where differences are
Then generate general hypotheses
This method generates lots of new data

Question 12

What is the bottom-up approach for metagenomics?

Answer 12

Work out novel function for a novel protein
If it’s in an environmental bacteria, you want to know what this protein is doing in the environment, its distribution, whether it associates with any environmental niches etc.
Can recycle available data in public databases

Question 13

What can primer bias vs shotgun approaches give?

Answer 13

Variations in data with areas of overlap but also areas of abundance where they are more enriched in either metagenome or 16S

Question 14

What was TARA Oceans and what meta- methods did it utilise?
What did all this data help us do?

Answer 14

Huge sampling effort of the oceans using metagenomic (DNA) and metatranscriptomics (RNA) data

Allowed us to understand how the oceans are working oin a molecular level

Question 15

What did the metagenomic data from TARA lead to?

Answer 15

Creation of a freely available web tool; Ocean gene atlas

Question 16

What is the method used for assembling and quantifying gene abundance? (hint - 2 levels of assembly; Inter- and intra-site)

Answer 16

Assemble contigs and identify genes and then take all the small reads that made up the contig and map them back to the contig
- The more reads assigned to a contig/specific gene, the more abundant that contig/gene is in environment; Generates abundance profiles

Then for each site, you can look for a specific gene and see which site has more reads mapped to it to see its abundance at a site
- Can also compare different genes within 1 site and see which gene is more abundant

Question 17

How can the method/algorithm for database searching impact conclusions?

Answer 17

Can show show differing results e.g. One gene being more distributed or abundant than other when they other method finds the opposite

Question 18

Explain Basic Local Alignment Search Tool (BLAST) (hint - E)

Answer 18

Set a stringency (cut-off); Expect (E) value – Lower E means better match
Position by position comparison
- Does 1:1 matching of query protein sequence
Insertions and deletions reduce score (not E)

Question 19

Explain Profile Hidden Markov Modelling (pHMM) in relation to BLAST
Using pHMM more PhoA hits were found than using BLAST. What does this say about BLAST? (hint - false)

Answer 19

Takes regions of conservation of protein into account; Can be more confident
Same stringency as BLAST search (e-60)

BLAST alone is not sufficient as it doesn’t fully uncover diversity
- False positives and/or false negatives

Question 20

Functional genes for P cycling? (4 genes)

Answer 20

phoX
phoD
phoA
glpQ

Question 21

How does abundance of PhoX and other phylogeny differ across regions and sites?

Answer 21

Their abundance varies across regions and sites

Question 22

Explain STrain Resolution ON assembly Graphs (STRONG)?

Answer 22

Assembly method allowing distinguishing of different strains
- Relies on building contigs
- Iterative approach
Get short reads from sequencing and co-assemble reads via algorithms to make contigs; Then map them to build up the genome and resolve different strain genomes

Question 23

How do phosphonates differ to most organic phosphorous bonds?

Answer 23

Phosphonate bonds are C-P which are stronger so they need specialised enzymes to be broken down

Question 24

What molecules can C-P lyase break down?
What does it have associated? (hint - transporter)
What is meant by it being promiscuous?

Answer 24

Phosphonates (C-P bond)

Has associated ABC transporter

Promiscuous; Works on multiple different substrates (phosphonates)

Question 25

What is an oligotroph?
Are they the least abundant group of organisms on the planet?

Answer 25

Oligotrophs (adapted to low nutrient environments)
No - Most abundant group of organisms on the planet

Question 26

What is the marine methane paradox?
How does methyl phosphonate breakdown link? (hint - limited phosphorous)

Answer 26

Large amounts of methane coming from oxygenated waters; Confusing as methanogenesis is an anoxygenic process

In regions of the ocean where inorganic Pi is limited, bacteria (e.g. SAR11) switch to breaking down methyl phosphonate; More methane production which is bad
In regions of the ocean where phosphorous is available, they don’t touch methyl phosphonate

Question 27

Why do some bacteria have 2AEP degradation pathways?

Answer 27

Microbes can degrade 2AEP as a sole nitrogen source

Question 28

What did metagenomics and MAGs enable? (hint - phosphonate)

Answer 28

Identification of many putative (predicted) phosphonate catabolism genes located near phosphonate transporters

Question 29

What does metagenomics only provide a snapshot of and how can metaproteomics help?

Answer 29

Metagenomics only gets a snapshot of genetic potential
Metaproteomics helps link structure and function

Question 30

When is metagenomics useful?
Metatranscriptomics can take us a step further through what?
Why is metaproteomics better than these

Answer 30

Analysing long-term shift in microbiome composition and function

Analysing how post-transcriptional regulation occurs in bacteria

Investigates the ‘functional entities’ of cell (protein); Machinery that drives reactions and causes effects

Question 31

Difficulties with metatranscriptomics and metaproteomics? (1 for each)

Answer 31

RNA is hard to handle, as once you sample bacteria their environment changes, so they change their transcriptome

Large computational requirements for metaproteomics

Question 32

Using metaproteomics what can we identify? (hint - nutrient stress)

Answer 32

Identify proteins (corresponding genes) that are induced under different nutrient stress conditions
e.g. Phosphatases

Question 33

What is bulk soil and rhizosphere?
Difference in metagenomic and metaproteomic profiles?

Answer 33

Bulk soil - Soil away from plant roots
Rhizosphere - Thin soil layer around plant root

So large difference in their metagenomic profiles but huge separation in metaproteomic profiles

Question 34

How would a rhizosphere adapted bacteria such Pseudomonas spp. be adapted to this environment?
Key trait of bulk soil adapted bacteria?

Answer 34

Produce rhizosphere specialist (plant) proteins with more pathways relating to P cycling

More oligotrophic due to low nutrient availability Can use a wider variety of nutrients

Question 35

When else is meta proteomics useful? (hint - growth)
Give an example

Answer 35

Identify pathways enriched during growth on various compounds e.g. complex polysaccharides which require many enzymes to hydrolyse each type of bond

Question 36

Using microbiota inside anaerobic bioreactors, what can we identify? Give an example

Answer 36

Distinct proteins like CAZymes which break down sugars

Question 37

What is the cause of the large computational cost of metaproteomics? (hint - data)

Answer 37

Combination of 2 datasets - DNA sequencing data with mass spectrometry data

Question 38

Problems with using mass spec. in metaproteomics?

Answer 38

Couldn’t separate all the peptide so we didn’t sequence all the peptides

Mass spec. doesn’t tell us everything about what peptide it is; Just m/z ratios