Microbial Genomics

Question 1

Genome

Answer 1

entire complement of genetic info including genes, regulatory sequences and noncoding DNA

Question 2

Genomics

Answer 2

discipline of mapping, sequencing, analyzing and comparing genes

Question 3

Sanger Dideoxy Sequencing Method

Answer 3

• uses radioactively labeled bases in PCR
• uses gel electrophoresis to sequence the DNA

Question 4

Illumina Sequencing Method

Answer 4

• 200 million reads/run; 300 bp length
• Steps:
  
  • put on double stranded adapters
  • have complimentary strands on flowell (600 million clusters)
  • creates build up/bridges on flowell
    
    • bridge amplification, stationary phase PCR
  • clusters are identified w/ fluorescence

Question 5

PacBio Sequencing

Answer 5

capable of 30,000 reads/run; 2000 bp length

Question 6

454 Sequencing

Answer 6

• 1 million reads/run; 700 bp length
• generates data 100x faster than Sanger method
• relies on 2 major advances
  
  • use of robotics
  • pyrosequencing
• Steps:
  
  • chop up DNA, attach things w/ ligase, undergo emulsion PCR which attaches DNA to bead
  • everything placed in picotiter plate (1.7 million wells)
  • nucleotides are run over DNA and will attach
    
    • releases PPi which get transferred to ATP which then admits light
  • light identifiable for each base

Question 7

How do you assemble the information from sequencing into a genome?

Answer 7

• align all reads w/ software to generate longer sequencing (contigs)
• gaps remain btwn contigs when don’t match up = “draft” genomes
  
  • orientation can be assumed if know related organisms
  • can amplify gaps by sequencing PCR amplicons
• most laborous and expensive part of sequencing

Question 8

Annotation

Answer 8

• coverting raw sequence data into a list of genes present in genome
  
  • majority of genes encode mRNAs (open reading frames)
  • some encode functional RNAs (rRNAs, tRNAs, ncRNAs)
• Functional ORF = Open Reading Frame (encodes proteins)
  
  • computers search for start/stop codons and shine-dalgarno sequences
    
    • identify ORF

Question 9

What is the correlation of gene size to genome size?

Answer 9

• gene = 1,000 bp
• 1,000 genes = 1 genome
• 1 Mbp = 1,000 genes = 1,000,000 bp

Question 10

What is the fractino of noncoding DNA in microbes?

Answer 10

• Eukaryotic microbes = >80%
• Bacteria = <15%

Question 11

How many kbp does Nanoarcheaum equitans have?

Answer 11

139 kbp

Question 12

How many kbp does *Mucobacterium tuberculosis *have?

Answer 12

4,400 kbp

Question 13

What is the estimated minimum # of genes needed for cell viability?

Answer 13

250 - 400 genes

Question 14

Which prokaryote has the larges genome to date at 13.0 Mbp?

Answer 14

Sorangium cellulosm

Question 15

Do archaeal genomes tend to be smaller or larger than bacterial ones?

Answer 15

smaller at only an estimated ~5 Mbp

Question 16

What is the general gene distribution in prokaryotes?

Answer 16

• the most abundant class is metabolic genes
• a minor class is made up of DNA replication and transcription genes
• prevalent class is those genes for nontranslated RNAs (rRNA, tRNA, ncRNA)

Question 17

Compare gene distribution between Bacteria and Archaea:

Answer 17

• archaea devote higher % to energy and coenzyme production
• bacteria devote higher % to carbohydrate metabolism and cell membrane fxn

Question 18

Metagenome

Answer 18

• total gene content of the organisms present in an environment
• usually used to analyze all of 16S genes, and then compare to determine present organisms

Question 19

Transcriptome

Answer 19

• the entire complement of RNA produced under a given set of conditions

Question 20

Why are Transcriptomes studied?

Answer 20

• to analyze …
  
  • global gene expression
  • expression of specific groups of genes under diff conditions
  • expression of genes w/ unknown fxn; can yield clues to its role
  • comparison of gene content in closely related organisms
  • ID of specific organisms

Question 21

How is the Transcriptome measured?

Answer 21

• qRT-PCR = Qualitative Real Time - PCR (only for individual genes)
• Microarrays (for whole transcriptomes)
• RNA-seq (for whole transcriptomes)

Question 22

Microarrays

Answer 22

• small, solid supports to which genes or segments of genes are fixed and arrayed spatially in known pattern
• gives a relative estimation of RNA abundance btwn 2 conditions

Question 23

Steps of using Microarrays:

Answer 23

1. isolate RNA
2. cDNA generation w/ PCR
3. labeling of the diff samples
4. hybridization of microarray (indicates transcribed gene)
5. imaging
   
   1. when A>B
   2. when B>A
   3. when A=B

Question 24

RNA-seq

Answer 24

• used to compare 2 conditions
• collect all RNA from sample
• convert to cDNA
• sequence w/ nex-gen
• align sequences to genome
• count reads for each gene
• compare samples

Question 25

Proteomics

Answer 25

* study of all the proteins that are present in a cell under a given condition * what DOES get made * more informative * measure by: 2D-gels, Mass spec

Question 26

2D-Gels

Answer 26

* techniqe for the separation, idenfication, and measurement of all proteins present in sample * Steps: * collect proteins * digest w/ trypsin (protease) * ionize them * pump through mass spec * match peptide fragments w/ ORF's predicted from genome * compare samples for relative abundance

Question 27

Interactome

Answer 27

* interaction maps that look at how all genes/proteins in cells react together

Question 28

Metabolomics

Answer 28

* the complete set of metabolic intermediates and other small molecules produced in an organism * techniques: mass spec

Question 29

Paralogs

Answer 29

genes w/in an organism whose similarity to one or more genes IN THE SAME ORGANISM is the result of gene duplication

Question 30

Orthologs

Answer 30

genes found in one organism that are similar to those IN ANOTHER ORGANISM but differ b/c of speciation

Question 31

Xenologs

Answer 31

gene duplications that arise due to the result of horizontal gene transfer

Question 32

Core Genome

Answer 32

genes shared by all strains of the species

Question 33

Pan Genome

Answer 33

includes all the optional extras present in some but not all strains of the species