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Microbiota On Mucosal > LA3- Genomics > Flashcards

LA3- Genomics Flashcards

1
Q

What did they use first for single genome analysis

A

Isolates cultured

Then panfenome sequencing of strains eg e0157 stx ecoli

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2
Q

What is metagenomics

A

Random sequencing of whole popn with a mix of species in a habitat eg the gut

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3
Q

What does this bypass need for

A

Isolation which has culture bias

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4
Q

What are the steps of wgs of communities (meta genome)

A

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

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5
Q

What would you use to find the microbes present

A

16s rRNA amplicon
Amplified with PCR and sequenced

Bioinformatics used to align sequences , and do phylogenetic analysis

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6
Q

What regions of the 16s RRNA will differ between species so can be determined via databases

A

Hyper variable regions

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7
Q

What is OTU

A

Organisms with 97% homology

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8
Q

For the question whag are they doing? What would you do

A

Shot gun wgs

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9
Q

What was used after Robert coch cultivation late 1800s

A

Amplicon

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10
Q

What was one of the earliest sequencing techniques used for amplicon and what was the advantage

A

Sanger sequencing
(Dideoxy)
Produced long reads meaning you can get species and genus level due to more hyper variable info

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11
Q

Give another with long reads that then replaced Sanger because it was too expensive

A

Roche purosequencing

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12
Q

Why did illumina sequencing replace these and what would be an issue theoretically but isn’t because of it being high throughput

A

Low cost and larger sample depth (could run more at once)

Shorter reads as it looks at 1 hyper variable region only

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13
Q

What does info from HMP do

A

Produce a reference catalog of genomes from cultures

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14
Q

After illumina hiseq used for metagenomics, what tool is used to run the sequence against databases to identify protein coding genes

A

Blast search

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15
Q

Give 2 examples of functional databases and what they do

A

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

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16
Q

How are these databases still biased

A

Biased to functional genes and cultured genomes

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17
Q

What 3 types of study designs are used

A

Cross sectional - all popn at one time

Longitudinal - repeated observations/samples

Case-control

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18
Q

What allows infants to be colonised

A

Down reg of tlr

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19
Q

Fr- why would preweaning bifidobacterium be most common

A

Use lacto-n-neotetraose HMO whcih can’t be used by others eg btheta

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20
Q

Give an example of how baxteroidetes and frimicutes can fluctuate in adulthood

A

Obesity ob/ob mouse shown firmicutes increased in ratio

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21
Q

Looking at databases like KEGG what other genes were enriched

A

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

22
Q

Which archaea was enriched in taxonomy aswell as firmicutes shown by pyrosequencing and why is this relevant

A

Methanobrevibacter smithii

It is a methanogrn, consumes h2 for better fermention

23
Q

What do all these enrhciehd pathways and Microbiota suggest in obese models

A

Better at harvesting energy = energy imbalance

24
Q

What’s the difference between microbial eukaryote and bacterial abundance in mega genome analysis

A

Fluctuation/heterogeneity in eukaryotes eg some will have blastocystis presence and some won’t

The bacterial ratio between B and F is consistent usually

25
Q

What do cazy databases cover

A

Major cazyme families like gh, pl, carb esterases

26
Q

Using 177 reference bac genomes annotated in the CAZY database, how many gh were present vs human

A

9120 gh vs 17 human

27
Q

Aswell as complex glycans from diet, how can mucin be a source

A

Has o-linked glycans attached

28
Q

Why can bacteroidetes use hmo eg b theta

A

Similar structure to o-linked glycan mucins

29
Q

Why is human milk a prebiotic

A

Provides glycans which can’t be degraded by us but can colonise other bacteria

30
Q

Which bacteria act synergistically with bifido which degrade oligosaccharide then provide hm monosach

A

Lactobacillus (not in formula milk)

31
Q

Give an example of bacteroidetes intraspecies preferences because of pul enzymes

A

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

32
Q

What do puls encode

A

Glycan binding and import proteins alongside surface and periplasmic cazymes

33
Q

How many puls do bt have for specific glycans

A

88

34
Q

How many are for o-linked mucin glycans

A

15

35
Q

Give 1 pul example on bt

A

Pul 4240-50

36
Q

The protein bt4244 in this pul from bt was found to have which newly identified domain after sequence alignment

A

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)

37
Q

The run on proteins containing pfam13402 metallopeptidase domain contained what whcih suggested they were for mucin utilisation

A

Tmd suggesting it works cell surface

And glycan binding domain like CBM32 and bacon (in bt4244)

38
Q

Which other species had this new pfam13402 domain

A

Others like akkermansia muciniphila (mucin using)
And also trichomonas vaginalis has pfam13402 - mucin degrading?

39
Q

What does the final hypothetical model look like for this bt4244 metallopeptidase containing protein/o-glycan peptidase in pul4240-50

A

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

40
Q

What did martens find in vivo and in vitro about bt4244 in presence of mucin

A

Upregulated expression

41
Q

Why is pul4240-50 suggested to be important for the colonisation

A

73% of the human metagenome encodes 1 or more of the pul4240-50 from bt, caccae or faecis

42
Q

Explain the study of desai 2016

A

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

43
Q

What transcripts were upreg in ff

A

Mucin-o glycan utilising proteins like pfam13402 containing

Also the cazymes for mucin

44
Q

What happened to the colon mucus barrier in ff

A

Was degraded and citrobacter rodentium colonised

45
Q

How many genes in ff of b caccae were enriched

A

14 genes

46
Q

What evidence was gathered to show this new domain pf13402 has Mucinase activity hypothesised from the bioinformatics info eg about having cbm and tmd

A

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

47
Q

How is the genome reassembled by fragments

A

Algorithm Look for overlap in dna sequences

48
Q

Why would you align contigs with a reference database

A

To identify based on taxonomic level

49
Q

When did the hmp begin and achieve

A

2007-

50
Q

Which endo-alpha sialidase do Ibd specific ecoli strains possess when compared with healthy metagenomics data - enriched sialyl tn ag = selective pressure

A

Gh58

51
Q

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

A

Sus c and d

52
Q

Did desai find the enzyme with pfam 13402 (m60 metallopeptidase) upreg in transcript in low fibre

A

Yes!

Microbiota On Mucosal (22 decks)

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