genome annotation

Question 1

annotation

Answer 1

• describing features of raw sequence:
  
  • genes
  • regulatory features
  • repeats
  • areas of conservation
  • syntenic regions
    
    • evolutionary relationships between genomes

Question 2

process of annotation

Answer 2

• assign gene functions
  
  • includes variable splice site identification
• identify regulatory features and functions
  
  • can be subtle
• identify repeats
• comparative studies with other genomes
• mainly automated but also VEGA manual intervention in places
• context is important

Question 3

simple metrics

Answer 3

• first step
• counting and analysing structure
• includes base confidence scores
• rolling GC content
• di/trinucleotide bias
  
  • patterns present more often than expected by chance → role
• codon use
  
  • increased use → common tRNAs → gene more easily expressed
  • gene with rare codons expressed less
  • housekeping genes → more common codons

Question 4

GC content

Answer 4

• important for gene prediction
• influences survival in the environment
• varies widely across genome and within genomes
• correlation between exon length and gene content
  
  • much higher GC with shorter introns
  • 65% (300 base) vs 30% (2300 base)

Question 5

human GC content

Answer 5

• ~38%, varies widely
• between 35% and 60% in a 100kB fragment
• in genes:
  
  • more uniform
  • 45-50%
• regions of high GC generally higher gene density
  
  • software based prediction

Question 6

bacterial replication

Answer 6

• oriC on both strands → replication in both directions
• reverse strand delayed due to opening and okazaki fragments
• ssDNA exposed → deamination of C to T 100x more frequently
• TG mismatch → mutate to TA in next round of replication
  
  • loss of C on reverse strand
  • relative decrease in G on reverse strand
  • decrease in C on forward strand
• plot skew
  
  • minimum - origin
  • maximum - termination

Question 7

GC skew in higher organisms

Answer 7

• multiple origins of replication
• 3 minima indicates 3 oriC e.g. archaea
• more difficult in eukaryotes
  
  • yeast - 400 ori
  • need to look for other patterns (ARS)

Question 8

eukaryotic GC skew

Answer 8

• ARS consensus sequence in yeast

Question 9

other measures

Answer 9

• dicodon counts
  
  • frequency of occurrence of successive codon pairs
• 3rd base periodicity
  
  • e.g. same nucleotide at n, n+2 etc
• length and occurrence of ORFs
  
  • stretches between stop codons
• promoters and TF binding sites (subtle)

Question 10

subtlety in human genome

Answer 10

• G and C more prevalent in first 50 nt of intron
  
  • GGG 4x more frequent
• VWG consensus in exons
  
  • not T, A/T, G
  • minimal periodicity of 10 nt
  • weaker in introns
  • phase bending potential towards major groove
    
    • increased accessibility