microbial genomics

Question 1

what is human genome projecT?

Answer 1

1990 - goal to sequence al 3*10^9 base pairs of human DNA

- completed in 2003 - thought it would take 30 year.s

Question 2

who did HGP?

Answer 2

public: US govt, Francis Collin
private: The institute Craig venter

Question 3

first completed genome sequence was?

Answer 3

haemophilus influenzae

- respiratory disease. took 8 years thru private funding

Question 4

venter style?

Answer 4

• break dna into pieces, sequence all at same time. computer sees where overlap + sticks together where overlaps.
  = contigs : overlap
  = less accurate bc gaps btw regions + repetitive sequences.
  but cheap + fast

Question 5

collins style?

Answer 5

simplistic approach
- sequence DNA. find end of gene. create probe that extended from end of piece to start of next piece

slow, systematic. had to create probe between each finding.

Question 6

sequencing DNA

Answer 6

sequence thousands of clones at one. 7-10 times coverage of entire genome

• assembles contigs using computer algorithms
• fill in gaps using targeted methods - chromosomal walking

Question 7

what’s sanger sequencing?

Answer 7

DNA polymerase in soln copies after primer.

di-deoxynucleotide (no -OH) addd to soln. DNApol can’t copy if no -OH.
separate based on size. know based on dideoxy what original nucleotide is.
shorter strands at beginning of sequence

Question 8

next gen sequencing

Answer 8

• increase in amount of genome/metagenome sequence. (many sequences at a time)
• non-specialist labs able to use (signal when nucleotides incorporated; quick, cheap)
• useful for resequencing (comparative - looking for disease)
• shotgun sequencing + computer power.

Question 9

structure of ORF

Answer 9

open reading frame
- approx 300 bp before stop codon.
ORF =/= gene. may be, but doesnt need to be. if transcribed + translated = gene.

Question 10

using ORF

Answer 10

1. comp finds codons
2. computer finds possible stop codons
3. combuter counts codons between start + stop.
4. computer finds possible RBS
5. computer calculares codon bias in ORF
6. computer decides if likely to be genuine
7. comp gives list of probably ORF

Question 11

ORF content to genome size

Answer 11

greater genome size = more ORF content

Question 12

lifestyles of bacteria

Answer 12

endosymbiotic - live in other cell. use host DNA, but have their own

parasitic: may grow inside cell. can’t grow without host DNA

Free-living: independent. dont need other organisms. usually larger than others. fitness cost

Question 13

gene annotation

Answer 13

compare ORF, if similar sequence annotated as similar function.
- gene annotations help reconstruct metabolic pathways + determine gene complement

Question 14

problem with gene annotation?

Answer 14

2 sequences may have diff function

- if dont have sequence similar, maybe protein is still same function

Question 15

pathogens + growth factors

Answer 15

from host cell.
no genes for amino acid biosynthesis
- no genes

Question 16

what is URF

Answer 16

ORF with unknown function

Question 17

Re-constructed Genome map

Answer 17

ORF’s in opposite directions.

Question 18

analyze map of bacteria + determine what kind of bacteria 1

Answer 18

• sugar transport
• peptide transport
• flagellum
• NH4+
  Zinc, Fe.

glycolysis, PPP
fermenter. lactate, no O2

Question 19

analyze map of bacteria + determine what kind of bacteria 2

Answer 19

TCA cycle. 
has ETC
flagella, 
pilli
have NADH oxidizer

Question 20

analyze map of bacteria + determine what kind of bacteria 3

Answer 20

calvin cycle, carbon fixation.
citric acid cycle
photosynthetic -
NH3 + O2 at 1st etc,

ammonia-oxidizing bacterium.
chemolithoautotroph
aerobe

Question 21

ORFs in bacterial genome : genome vs function

Answer 21

DNA replication: same amount needed regardless of genome. proportionally decrease tho. same with translation

trancription + signal transduction : more regulation, more pathways when there’s bigger size.

energy: about stable - increase genome doesnt change energy much

Question 22

what are homologous genes

Answer 22

from same ancestor

Question 23

diff btw paralog and ortholog

Answer 23

paralog: gene duplication: gene stays within same species or organism (a and b-heme)
- 1 gene retains function, other may vary/pseudogene.

2. ortholog: copies of same gene, split into diff species same function in 2 diff species.

Question 24

define synteny

Answer 24

similar gene order

- more closely related = more syntenous

Question 25

core genome vs acnillary genome vs pan genome

Answer 25

core: shared by all strains of same species (specific to group of interest) ancillary: foudn in some strains of same species (one's not common to all) pan: all genes found in group

Question 26

looking for HGT - look for?

Answer 26

gene islands: GC diff than rest of chromosome, codon usage bias. flanked by inverted repeats. transferred by HGT transposons: move around, inverted repeats. pathogenicity islands: turn normal into virulent factor block chromosomal genes that encode virulense

Question 27

what is metagenoics

Answer 27

focus on individual genes previously. | -all genes in enviro. extract DNA and reconstruct community

Question 28

ocean metagenomic analysis

Answer 28

look for DNA from cloned genes linked to rRNA gene to ID what organisms might be doing SR: abundant, couldnt culture.. found RNA gene in it + sequenced - related to rhosopsin. - uses light energy to pump Cl- out. when put that gene into e.coli - could pump H+ across using light. photoheterotrophs use to gain more energy from food they have. little nutrient in enviro, light gives energy

Question 29

human microbiome size

Answer 29

10-100 trillion bacteria mostly in gut. | connection btw human health + illness

Question 30

bacteroides and obesity

Answer 30

bacteroides + firmicutes = break down food. obese: less bacteroides + more firmicutes than slim ppl bacteroides increase as obese ppl lose weight

Question 31

diff btw bacteroides + firmicutes?

Answer 31

firmicutes get more energy out of food =

Question 32

sequence analysis: bioinformatics

Answer 32

``` computational analysis of genome data - genome content, structure, arrangement - predict protein structure + function - produce annotation to determine that location of genes (databases contain gene info) = in silico analysis. ```

Question 33

reverse transcriptase

Answer 33

enzyme from retrovirus used to produce DNA copies "cDNA" of their genome to integrate into host genome

Question 34

comparative genomics

Answer 34

look at groups of organisms relate to each other. | - ID the overlaps in genome. find core + ancillary genomes

Question 35

mutations in E.coli - evolutionary pressures

Answer 35

150000 generations + Id'd similarities.ID'd mutations in relation to ancestral condition

Question 36

Total mRNA

Answer 36

extracted from control + experimental cells to determine changes in gene expression

Question 37

creation of genomic microarray + creation of labeled probe representing all mRNA in cell at time

Answer 37

genomic DNA -> PCR -> probe hybridization -> signal detection -> data analysis probe hybridization: make cDNA porbe and label

Question 38

Microarraws: detecting differential gene expression

Answer 38

differently labeled cDNA from each culture mixed + hybridized to array. - labeled probe finds complementary sequence. hybridizes by normal DNA base-pairing rules - fluorescent signal detected where probe is hybridized. = require several replications and verigication by other methods

Question 39

Transcriptome of P.aeruginosa

Answer 39

- assemblage of microarray data | - global gene expression from cells grown wiwith hgih vs low levels of Ca2+

Question 40

RNa sequence

Answer 40

next gen - avoid microarray - sequences transciptome with rev.transcripted cDNA - quantitatively determine copy numbers of genes - tell what's up/down regulated in experimental relative to control

Question 41

what are proteomics?

Answer 41

dtermining differences in translation | = what proteins are being made?

Question 42

proteome?

Answer 42

proteins present in cell, tissue, or organism at any one point in time

Question 43

mRNA copies in glycolysis vs sporulation

Answer 43

glycolysis, some genes on, some off. sporulation - all increase diff regulation to diff levels

Question 44

proteomics interactome | single cell genomics

Answer 44

- > all proteins being produced in community. | - > looking at genome only within one cell