1. Pyrosequencing at LWH
Carly Broadhurst

2. Overview Principle of Pyrosequencing Sample preparation
Applications at LWH
Strengths of Pyrosequencing
Troubleshooting
Future of Pyrosequencing at LWH

3. 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.
DNA polymerase, ATP sulfurylase, luciferase and apyrase, and the substrates, adenosine 5´ phosphosulfate (APS) and luciferin. 
Each incorporation event is accompanied by release of pyrophosphate (PPi) in a quantity equimolar to the amount of incorporated nucleotide.

4. 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.
ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5´ phosphosulfate. This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. 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.

5. 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.  

6. Process of events PCR Sample preparation Pyrosequencing
PCR – primers flank region of interest

7. Primers
Red circle = biotinylated primer (biotage)

8. 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.   

9. Vacuum Prep Worktable Streamlines the sample preparation process
Vacuum Prep Worktable Streamlines 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.

11. 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

12. 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

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

14. Absence/presence of mutation e.g. Haemachromatosis
n/n
p.C282Y G/G
p.C282Y G/A
M/n

15. p.C282Y A/A
M/M

16. 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)

17. 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)

18. 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.

19. 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)