sequencing & assembly

Question 1

stages of genome sequencing

Answer 1

• fragmentation/cloning
  
  • fragment library, amplification
• sequencing
  
  • from both ends to get multiple reads
• processing
  
  • base calling, quality assessment, repeat masking
  • trim ends (decrease on polymerase affinity)
• assembly
  
  • overlapping reads → contigs
  • contigs → scaffolds

Question 2

pac bio

Answer 2

• 3rd gen
• longest reads ~20,000
• high error rate but random
  
  • mutliple reads → consensus
• 99.999% accuracy

Question 3

phred scores

Answer 3

• quality score
• estimated confidence in each base call
• use to:
  
  • filter and trim reads
  • create consensus
  • distinguish between variants and errors

Question 4

Q value

Answer 4

• given by phred
• QV = -10log10(Pe)
  
  • Pe = probability that base call is an error
• ignore call if lower than 30
  
  • <99.9% accuracy

Question 5

chastity filter

Answer 5

• illumina base call algorithm
• assign and filter intensity score for nucleotides at each position
• highest score divided by sum of highest and 2nd highest score for that position
• less than threshold (0.6) base marked N
• if higher assign base call

Question 6

factors affecting quality

Answer 6

• end of read deterioration (pol affinity)
• adaptor attached to reads
• high AT or GC content
  
  • reduced complexity
• homopolymeric tracts
  
  • unsure of length
  • SNPs ignored (assumed as error)

Question 7

depth of coverage

Answer 7

• eliminate errors
• depends on genome complexity, read length, sequencer error rate
• HGP - 12x or greater
  
  • each base present in 12 reads on average

Question 8

paired end sequencing

Answer 8

• sequence both fragment ends
• distance known → filter fragments by size
• knowing one position anchors the other
• better read alignment
• important for repeats
• improved prediction of structural variations

Question 9

repeats

Answer 9

• fragments with identical repeat regions can be assembled together
• in between sequences lost
• sequencing may be impossible

Question 10

repeats and paired end reads

Answer 10

• pair of overlapping reads, 1 unique, 1 repetitive
• map unique read
• position second as distance known
• enough paired reads allows sequencing across whole repeat region
• small repeats only

Question 11

mate pairs

Answer 11

• longer than paired ends
  
  • kb vs 500bp
• bridge across repeats or structural rearrangements
• don’t sequence repeat but don’t lose information
• fill gaps with paired ends
• helps resolve correct order of repeat fragments

Question 12

scaffolding

Answer 12

• resolution of conflicting areas
• order non-overlapping contigs into scaffolds
  
  • gaps with known or predicted size
  • spanned by N (unknown sequence)
• bridge contigs with mate pairs
• de novo assembly - gaps remain
  
  • need wet-lab work and paired end reads

Question 13

rearrangments and paried end reads

Answer 13

• compare to reference genome mapping
• decrease in size → deletion
• increase in size → insertion
• wrong way round → inversion
• maps to different region → translocation

Question 14

finishing

Answer 14

• fill in gaps, resequencing, different technology or longer reads
• design primer probe for PCR to reach end
• improve ocnsensus
• expensive

Question 15

limitations of assembly

Answer 15

• next gen small read lengths
• AT rich genomes
• repetitive genomes
• de novo sequencing
  
  • no reference
  • use multiple technologies

Question 16

entamoeba histolytica

Answer 16

• AT rich
• long runs of Ts
• many contig ends are T
• unknown ploidy
• 1500 contigs, 20Mb

Question 17

blumeria araminis

Answer 17

• repeat rich
• 7000 contigs, 120Mb
• contig assembly difficult
• need longer reads or targeted sequencing