LA3- Genomics Flashcards
What did they use first for single genome analysis
Isolates cultured
Then panfenome sequencing of strains eg e0157 stx ecoli
What is metagenomics
Random sequencing of whole popn with a mix of species in a habitat eg the gut
What does this bypass need for
Isolation which has culture bias
What are the steps of wgs of communities (meta genome)
Fragmentation of dna extracted
Computer algorithms assemble it into long reads called scaffolds
Sequence then annotated using information from databases like KEGG or CAZy for gene prediction
What would you use to find the microbes present
16s rRNA amplicon
Amplified with PCR and sequenced
Bioinformatics used to align sequences , and do phylogenetic analysis
What regions of the 16s RRNA will differ between species so can be determined via databases
Hyper variable regions
What is OTU
Organisms with 97% homology
For the question whag are they doing? What would you do
Shot gun wgs
What was used after Robert coch cultivation late 1800s
Amplicon
What was one of the earliest sequencing techniques used for amplicon and what was the advantage
Sanger sequencing
(Dideoxy)
Produced long reads meaning you can get species and genus level due to more hyper variable info
Give another with long reads that then replaced Sanger because it was too expensive
Roche purosequencing
Why did illumina sequencing replace these and what would be an issue theoretically but isn’t because of it being high throughput
Low cost and larger sample depth (could run more at once)
Shorter reads as it looks at 1 hyper variable region only
What does info from HMP do
Produce a reference catalog of genomes from cultures
After illumina hiseq used for metagenomics, what tool is used to run the sequence against databases to identify protein coding genes
Blast search
Give 2 examples of functional databases and what they do
KEGG and cazy
KEGG quantifies abundance of protein genes in a particular pathway eg carb metabolism
Cazy is a metabolic database with info on all enzymes
How are these databases still biased
Biased to functional genes and cultured genomes
What 3 types of study designs are used
Cross sectional - all popn at one time
Longitudinal - repeated observations/samples
Case-control
What allows infants to be colonised
Down reg of tlr
Fr- why would preweaning bifidobacterium be most common
Use lacto-n-neotetraose HMO whcih can’t be used by others eg btheta
Give an example of how baxteroidetes and frimicutes can fluctuate in adulthood
Obesity ob/ob mouse shown firmicutes increased in ratio
Looking at databases like KEGG what other genes were enriched
ABC transporters (firmicutes use), butanoate pathway proteins found abundant in KEGG database on the Ob ob mouse
Galactose metabolism etc
Cazy active firmicutes allowing utilisation of complex glycans = increased energy harvesting
Which archaea was enriched in taxonomy aswell as firmicutes shown by pyrosequencing and why is this relevant
Methanobrevibacter smithii
It is a methanogrn, consumes h2 for better fermention
What do all these enrhciehd pathways and Microbiota suggest in obese models
Better at harvesting energy = energy imbalance
What’s the difference between microbial eukaryote and bacterial abundance in mega genome analysis
Fluctuation/heterogeneity in eukaryotes eg some will have blastocystis presence and some won’t
The bacterial ratio between B and F is consistent usually
What do cazy databases cover
Major cazyme families like gh, pl, carb esterases
Using 177 reference bac genomes annotated in the CAZY database, how many gh were present vs human
9120 gh vs 17 human
Aswell as complex glycans from diet, how can mucin be a source
Has o-linked glycans attached
Why can bacteroidetes use hmo eg b theta
Similar structure to o-linked glycan mucins
Why is human milk a prebiotic
Provides glycans which can’t be degraded by us but can colonise other bacteria
Which bacteria act synergistically with bifido which degrade oligosaccharide then provide hm monosach
Lactobacillus (not in formula milk)
Give an example of bacteroidetes intraspecies preferences because of pul enzymes
The fructan levan used more by b theta because it contains gh32 hydrolysing only b2-6 fructans
Pl19 by ovatus and caccae puls prefer b2-1 fructan inulin
What do puls encode
Glycan binding and import proteins alongside surface and periplasmic cazymes
How many puls do bt have for specific glycans
88
How many are for o-linked mucin glycans
15
Give 1 pul example on bt
Pul 4240-50
The protein bt4244 in this pul from bt was found to have which newly identified domain after sequence alignment
Pfam 13402 which was a domain characteristic to zinc-m60 metalloproteinases motif (suggesting bt4244 is a peptidase) (also contains cbm32 and bacon for glycan binding)
The run on proteins containing pfam13402 metallopeptidase domain contained what whcih suggested they were for mucin utilisation
Tmd suggesting it works cell surface
And glycan binding domain like CBM32 and bacon (in bt4244)
Which other species had this new pfam13402 domain
Others like akkermansia muciniphila (mucin using)
And also trichomonas vaginalis has pfam13402 - mucin degrading?
What does the final hypothetical model look like for this bt4244 metallopeptidase containing protein/o-glycan peptidase in pul4240-50
It binds and cleaves the mucin gp backbone with the help of the sgbp (suse) (bt4245)
Before mucin glycan can be used and imported via sus c and d
Deglycosylation of the processed core peptide by gh2 and gh109 in this PUL eg de glycosylation of galnac
What did martens find in vivo and in vitro about bt4244 in presence of mucin
Upregulated expression
Why is pul4240-50 suggested to be important for the colonisation
73% of the human metagenome encodes 1 or more of the pul4240-50 from bt, caccae or faecis
Explain the study of desai 2016
Colonised germ free mice with annotated and sequenced genome bacteria like akkermansia muciniphila, b caccae
Given either ff or fibre rich diet
Transcriptomics and metagenomics used to detect taxonomy variation and gene exp variation
What transcripts were upreg in ff
Mucin-o glycan utilising proteins like pfam13402 containing
Also the cazymes for mucin
What happened to the colon mucus barrier in ff
Was degraded and citrobacter rodentium colonised
How many genes in ff of b caccae were enriched
14 genes
What evidence was gathered to show this new domain pf13402 has Mucinase activity hypothesised from the bioinformatics info eg about having cbm and tmd
Full length recombinant bt4244 protein was able to degrade the bovine mucin backbone, aswell as recombinant bt4244 with only the pf13402 domain
No degradation of mucin backbone in absence of the pf13402 domain
How is the genome reassembled by fragments
Algorithm Look for overlap in dna sequences
Why would you align contigs with a reference database
To identify based on taxonomic level
When did the hmp begin and achieve
2007-
Which endo-alpha sialidase do Ibd specific ecoli strains possess when compared with healthy metagenomics data - enriched sialyl tn ag = selective pressure
Gh58
Which 2 proteins in the sus system for o glycans by b theta (pul 4240-50) was found in the b theta strain adaptation to low fibre/westernised diet to have developed mutations
Sus c and d
Did desai find the enzyme with pfam 13402 (m60 metallopeptidase) upreg in transcript in low fibre
Yes!
