New Generation Sequencing

Question 1

What is the $1000 genome?

Answer 1

• cost for sequencing a human genome too high for most experiments
  
  • in 2004, 100,000,000
• US government offered grants to make DNA sequencing more cost effective (1000 per genome)

Question 2

Sanger DNA sequencing

Answer 2

• with fluorescent dyes
• start from primer
  
  • grow DNA chain
• uses dideoxynucleoside
  
  • modified bases so reaction stops
  • stopped at all possible points
• separate resulting sequences with gel electrophoresis
  
  • separation by lenght

Question 3

problems with Sanger DNA sequencing

Answer 3

• Sensitivity
  
  • several fragments necessary for signal, amplification
  • baterial cloning, bottleneck
• Electrophoresis
  
  • separation of the sequencing in individual electrophoresis
  • other bottleneck

Question 4

PCR

Answer 4

• no bacterial cloning
  
  • DNA fragments are connected with adapters and put in the library
• Emulsion PCR
• Bridge PCR

Question 5

NGS

Answer 5

• libraries based on PCR colonies
• different types of sequencing
  
  • 454, pyrosequencing
  • Illumina → Sequencing by synthesis (Sanger-like)
  • SOLiD → Sequencing by Ligation
  • single molecule sequencing

Question 6

Pyrosequencing

Answer 6

• based on sequencing by synthesis
• relies on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides
• DNA sequence determined by light emitted upon incorporation of the next complementary nucleotide
• previous nucleotide is degraded

Question 7

Illumina

Answer 7

• sequencing technology based on reversible dye-terminators
• DNA primed and amplified (bridge)
• reversible terminator bases are added (4)
• non-incorporated nucleotides are washed away
• one nucliotide at a time
• dye is chemically removed from the DNA, allowing the next cycle

Question 8

SOLiD ligation probes

Answer 8

• 3’ Ligation site, cleavage site & dye are spatially separated
• Ligation Probes are Octamers
• 2-base encoding in color space
• sequential rounds of sequencing, multiple cycles per round

Question 9

Single molecule

Answer 9

• Pacific Biosciences, PacBio
• nucleotides labelled with different colored fluorophore
• lightpulse produced

Question 10

Ion torrent

Answer 10

• direct connection between chemical and digital information
• simpler, faster, more cost effective and scalable
• previous had intermediary (light) to translate chemical data into digital data
• nucleotide incorporated -> hydrogen ion released
  
  • carries a charge, two identical bases, double voltage
  • one nucleotide at a time given

Question 11

Genia - nanopore

Answer 11

• $100 genome goal
• single DNA molecules
• reduces the price of sequencing and increases speed, accuracy, and sensitivity
• nanopore -> protein pore with lipid bilaye membrane
• DNA replication enzyme sequences a template strand with single base precision
  
  • base specific tags cleaved by enzyme are captured by nanopore