Pyrosequencing

Pyrosequencing is a method of DNA sequencing (determining the order of nucleotides in DNA) based on the "sequencing by synthesis" principle developed by Mostafa Ronaghi and Pål Nyrén (published in Analytical Biochemistry 1996 and Science 1998). For the history of the Pyrosequencing method see Methods Mol Biol. 2007. Pyrosequencing AB was started to commercialize the machine and reagent for sequencing of short stretches of DNA. Pyrosequencing AB was renamed to Biotage in 2003. Pyrosequencing technology was further licensed to 454 Life Sciences. 454 developed an array-based Pyrosequencing which has emerged as a rapid platform for large-scale DNA sequencing. Most notable are the applications for genome sequencing and metagenomics. GS FLX, the latest pyrosequencing platform by 454 Life Sciences (owned by Roche), can generate 100 million nucleotide data in a 7 hour run with a single machine. It is anticipated that the throughput would increase by 5-10 fold with the next release. Each run would cost about 8,000-9,000 USD, pushing de novo sequencing of mammalian genomes into the million dollar range.

Additional recommended knowledge

Essential Laboratory Skills Guide

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Procedure

The method is based on a chemical light-producing enzymatic reaction, which is triggered when a molecular recognition event occurs. Essentially, the method allows sequencing of a single strand of DNA by synthesizing the complementary strand along it. Each time a nucleotide, A, C, G or T is incorporated into the growing chain a cascade of enzymatic reactions is triggered which causes a light signal.

ssDNA template is hybridized to a sequencing primer and incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase and apyrase, and with the substrates adenosine 5´ phosphosulfate (APS) and luciferin.

1. The addition of one of the four deoxynucleotide triphosphates (dNTPs) initiates the second step. DNA polymerase incorporates the correct, complementary dNTPs onto the template. This incorporation releases pyrophosphate (PPi) stoichiometrically.
2. ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5´ phosphosulfate. This ATP acts as fuel to 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 this can be analyzed in a program. Each light signal is proportional to the number of nucleotides incorporated.
3. Unincorporated nucleotides and ATP are degraded by the apyrase, and the reaction can restart with another nucleotide.

Currently, a limitation of the method is that the lengths of individual reads of DNA sequence are in the neighborhood of 300-500 nucleotides, shorter than the 800-1000 obtainable with chain termination methods (e.g. Sanger sequencing). This can make the process of genome assembly more difficult, particularly for sequence containing a large amount of repetitive DNA. As of 2007, pyrosequencing is most commonly used for resequencing or sequencing of genomes for which the sequence of a close relative is already available.

The templates for pyrosequencing can be made both by solid phase template preparation (Streptavidin coated magnetic beads) and enzymatic template preparation (Apyrase+Exonuclease).

Recent advances

The technique has been commercialized and further developed by 454 Life Sciences Corporation in its GS 20 and GS FLX line of sequencing machines.

Use in Research

Bee Virus

In September 2007, 454 pyrosequencing was used in a study implicating Israel acute paralysis virus in honeybee Colony Collapse Disorder ^[1].

External links and references

• http://www.biotage.com
• http://www.genome.org/cgi/content/full/11/1/3
• http://www.454.com
• http://www.ncbi.nlm.nih.gov/sites/entrez
• http://www.mgrc.com.my/eLectureRonaghi.shtml
• http://www.pyrosequencing.com
• Ronaghi M. "Pyrosequencing sheds light on DNA sequencing." Genome Research 2001 Jan;11(1):3-11. PMID: 11156611.
• Ronaghi et al. Real-time DNA sequencing using detection of pyrophosphate release. Anal Biochem. 1996
• Ronaghi et al. A sequencing method based on real-time pyrophosphate. Science 1998
• Ronaghi et al. Improved performance of Pyrosequencing using single-stranded DNA-binding protein. Anal Biochem. 2000
• Elahi et al. Pyrosequencing: a tool for DNA sequencing analysis. Methods Mol Biol. 2004
• Gharizadeh et al. Long-read pyrosequencing using pure 2'-deoxyadenosine-5'-O'-(1-thiotriphosphate) Sp-isomer. Anal Biochem. 2002
• Fakhrai-Rad et al. Pyrosequencing: an accurate detection platform for single nucleotide polymorphisms. Hum Mutat. 2002
• Nyrén The History of Pyrosequencing. Methods Mol Biol. 2007;373:1-14

References