Pyrosequencing at LWH

Carly Broadhurst

Overview

Principle of PyrosequencingSample preparationApplications at LWHStrengths of PyrosequencingTroubleshootingFuture of Pyrosequencing at LWH

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.

Principle of Pyrosequencing

The light produced in the luciferase-catalyzed reaction is detected by a charge coupled device (CCD) camera and seen as a peak in a pyrogram™. Each light signal is proportional to the number of nucleotides incorporated.

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.  

Process of events

PCR

Sample preparation

Pyrosequencing

PCR – primers flank region of interest

Primers

Red circle = biotinylated primer (biotage)

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.   

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.

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

Pyrosequencing applications at LWH

Haemachromatosis – p.C282Y + p.H63D (Absence/presence of mutation)

Quantification of mitochondrial DNA mutations: MELAS m.3243A>G MERRF m.8344A>G NARP/Leigh m.8993 T>C/G LHON m.11778G>A, m.3460G>A, m.14484T>C Deafness associated SNP m.1555A>G

Hereditary Pancreatitis – SPINK1 p.N34S mutation

110

120

130

140

150

160

E S T A G T C A C A C

A:100.0%G:0.0%

5

110

120

130

140

150

160

170

E S T A G T C A C A C

A:18.8%G:81.2%

5

m.8344A>Gm.8344A>G normalnormal

m.8344A>G m.8344A>G

heteroplasmicheteroplasmic

Quantification of mutation e.g. MERRF m.8344A>G

p.C282Y G/A

n/np.C282Y G/G

M/n

Absence/presence of mutation e.g. Haemachromatosis

p.C282Y A/A M/M

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 digestQuantification – important for

mitochondrial mutations (previously used MS-PCR end-point PCR)

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)

Troubleshooting

Hedgehog capturing beads - PCR plate needs to be held on a steady platform.

Reagent cartridge, don’t 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.

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)