DNA Sequencing

Process of Sanger Sequencing

Advantages & limitations of sanger sequencing

Compare & contrast sanger & pyrosequencing

Describe multiple approaches to next-gen sequencing

Provide examples of how NGS can be used to identify disease genes

Compare & contrast low & high throughput sequencing

Sanger Technique

Uses dideoxynucleotides

Molecules that resemble normal nucleotides but lack normal -OH group

Steps

1. DNA template denatured to single strands

2. Single DNA primer (3' end near sequence of interest) annealed to template DNA & extended with DNA polymerase

  1. Four reactions set up, each containing: DNA template, Primer annealed to template DNA, DNA polymerase, dNTPs (dATP, dTTP, dCTP, and dGTP)
  1. A different labelled dideoxynucleotide (ddATP, ddTTP, ddCTP, or ddGTP) added to each of four reaction tubes
  1. ddNTPs possess a 3' -H instead of 3' -OH, compete in reaction with normal dNTPs, & produce no phosphodiester bond
  1. Whenever labelled ddNTPs are incorporated in chain, DNA synthesis terminates
  1. Dideoxy DNA sequencing (dye terminator sequencing)
  1. Each of four reaction mixtures produces a population of DNA molecules with DNA chains terminating at all possible positions
  1. Extension products in each of four reaction mixtures also end with a different labelled ddNTP (depending on base)
  1. Each reaction mixture electrophoresed in a separate lane (4 lanes) at high voltage on a polyacrylamide gel
  1. Polyacrylamide gels can be thinner --> higher voltage --> faster
  1. Pattern of bands in each of 4 lanes is visualised on X-ray film or automated sequencer

Process of Pyrosequencing

Based on "sequencing by synthesis" principle instead of chain termination with dideoxy nucleotides

  1. Immobilise a single template DNA molecule on a bead/substrate & synthesise complementary strand
  1. Detect which nucleotide is added at each step. Sequencing (polymerisation) doesn't stop
  1. Requires template DNA, primer, DNA polymerase, ATP sulfurylase, luciferase, apyrase, adenosine 5' phosphosulfate (APS), and luciferin
  1. As with dideoxy sequencing, base incorporation recorded when light emitted at particular wavelengths

Sanger sequencing

Relies on electrophoretic separation of end-stage PCR products

Physical limits on number of capillaries

Relatively expensive

Pyrosequencing

Very expensive

Not easy to run many reactions at once

NGS

Steps

  1. Fragment target DNA & ligate universal adaptors
  1. Amplify single molecules (beads vs free)
  1. Sequence clonal amplicons
  1. Computer assemble the data

Each system differs in the way DNA is sequenced

Reference-based assemblies

De novo assemblies