Pyrosequencing at LWH

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Pyrosequencing at LWH

• Carly Broadhurst

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Overview

• Principle of Pyrosequencing
• Sample preparation
• Applications at LWH
• Strengths of Pyrosequencing
• Troubleshooting
• Future of Pyrosequencing at LWH

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Principle of Pyrosequencing

• Sequencing primer is hybridized to a single
  stranded, PCR amplified, DNA template and
  incubated with enzymes.
• The first of four dNTPs is added to the reaction.
  DNA polymerase catalyzes the incorporation of the
  dNTP into the DNA strand.

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Principle of Pyrosequencing
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Principle of Pyrosequencing continued . .

• Apyrase, a nucleotide degrading enzyme,
  continuously degrades unincorporated dNTPs and
  excess ATP. When degradation is complete, another
  dNTP is added.
•  
• As the process continues, the complementary DNA
  strand is built up and the nucleotide sequence is
  determined from the signal peak in the pyrogram.
   

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Process of events

• PCR
• Sample preparation
• Pyrosequencing

• PCR primers flank region of interest

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Primers
Red circle biotinylated primer (biotage)
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Sample preparation

• The Vacuum Prep Workstation -process up to 96 DNA
  samples in parallel, from PCR-products to
  single-stranded sequencing templates, in less
  than 15 minutes.
• Fast and efficient workflow is achieved due to
  the minimized use of pipetting and placement of
  the solution troughs.
• Hands-on time using the Vacuum Prep Tool is
  less than one minute.   

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Vacuum Prep WorktableStreamlines the sample
preparation process. It accommodates five troughs
for the different solutions necessary to process
the samples. One of the plate positions is
specifically designed to fit most commercially
available PCR-plates.
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Pyrosequencing

• Prepare cartridge
• Nucleotides
• Enzyme mix (DNA polymerase, sulphyrase,
  luciferase, apyrase)
• Substrate mix (luciferin, adenosine 5
  phosphosulfate (APS))
• Load cartridge annealing plate into machine
  ready to go!!!
• need file to tell the machine what the sequence
  of interest is so that it can determine the
  dispensation order of the nucleotide

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Pyrosequencing applications at LWH

• Haemachromatosis p.C282Y p.H63D
  (Absence/presence of mutation)
• Quantification of mitochondrial DNA mutations
• MELAS m.3243AgtG
• MERRF m.8344AgtG
• NARP/Leigh m.8993 TgtC/G
• LHON m.11778GgtA, m.3460GgtA, m.14484TgtC
• Deafness associated SNP m.1555AgtG
• Hereditary Pancreatitis SPINK1 p.N34S mutation

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Quantification of mutation e.g. MERRF m.8344AgtG
m.8344AgtG normal
m.8344AgtG heteroplasmic
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Absence/presence of mutation e.g.
Haemachromatosis
p.C282Y G/A
M/n
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p.C282Y A/A
M/M
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Strengths of Pyrosequencing

• Delivers the gold standard of genetic analysis
  the sequence itself
• Rapid (hands on time 1hour running samples on
  machine 10mins)
• E.g. Haemachromatosis mutations previously tested
  using PCR restriction digest
• Quantification important for mitochondrial
  mutations (previously used MS-PCR end-point PCR)

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Strengths of Pyrosequencing continued . .

• Genotyping straight forward - pyrograms easy to
  interpret
• Machine vacuum prep station - little
  maintenance
• Can take pyrosequencing plate back through the
  vacuum prep station (add binding buffer to plate)

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Troubleshooting

• Hedgehog capturing beads - PCR plate needs to be
  held on a steady platform.
• Reagent cartridge, dont soak overnight, rinse
  with warm water and run through with distilled.
  Replace cap and leave to dry upside down. Shelf
  life approx 25 30 runs.
• Slow probes on hedgehog (can be replaced quickly
  and easily).
• Denaturation solution (0.2M NaOH) needs to be
  made up regularly.

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Future of Pyrosequencing at LWH

• Automation - possibility of moving part of the
  process preparation of bead plate annealing
  plate to robot (particularly HCT as we receive
  lots of samples)