DNA Sequencing

A molecular technique to reveal the arrangement of nucleotides in polynucleotide chains...

Second Generation

Pyrosequencing

  • Principle described by Bertil Pettersson, Mathias Uhlen and Pål Nyren
  • Sequence-by-Synthesis (SBS)
  • Based on the detection of luminescence produced by the co-activity of 3 enzymes
    • DNA Polymerase
    • ATP Sulfurylase
    • Luciferase
    • Apyrase (Post-sequencing activity)
  • Microfluidic variant developed by Jonathan Rothberg (454 Life Sciences)
  • Output Format: SFF
  • Read length: 300-500 bases
Pyrosequencing Method

Short Read Sequencing (Illumina)

  • Concept invented by Bruno Canard and Simon Sarfati (Pasteur Institute, Paris)
  • Developed by Shankar Balasubramanian and David Klenerman (Solexa)
  • Reversible dye-terminators
  • Sequence-by-Synthesis (SBS)
  • Genomic Library Preparation - Sonification or Tagmentation (Transposases) - 50 to 500 bp fragments
  • Adapters have 3 parts - Adapter sequence, Index sequence, and Primer sequence
  • Output format: fastq
  • Read length: 35 to 300 bp
  • Instruments: GA, GAII, MiSeq, HiSeq, NovaSeq, etc...

Flow-cell

Illumina Sequencing Method

Third Generation

Single Molecule Sequencing (SMS) by Stephen Quake, commercialized by Helicos Biosciences - Similar to Illumina sequencing, excluding the bridge amplification step. First technology to sequence non-amplified DNA thus avoiding the amplification associated biases an errors. Slow and expensive

Single Molecule Real Time (SMRT) Sequencing (PacBio)

  • Hairpin adaptors
  • Phospholinked nucleotides with fluorophores
  • SMRTbell library
  • Zero-Mode Waveguides (ZMWs)
  • Continuous Long Read Sequencing (CLR)
  • Circular Consensus Sequencing (CSS)
  • Average Read Length: 10,000 bp - 100,000 bp
  • Instruments: RS, RS II, Sequel
  • Output format: BAM
Terminology
SMRT Sequencing Method

Nanopore Sequencing (Oxford)

  • Idea proposed by David Deamer in 1989
  • Hagan Bayley co-founded Oxford Nanopore in 2005
  • Uses Biological Nanopores like α-hemolysin, MspA, CsgG
  • Synthetic membranes
  • Submerged in ionic solution
  • Single strand from a dsDNA molecule is fed into the nanopore with the help of motor protein
  • Variations in the ionic current while incorporating different nucleotides is captured
  • Read Length of ~1 Mbp can be achieved
  • Instruments: MinION, PromethION, etc...
  • Output format: FAST5
Nanopore Sequencing Method