The Next-Generation Chemistry for Pyrosequencing

Pyro Gold is the new chemistry formulation for Pyrosequencing. The result of 2 years of research and development, Pyro Gold contains a number of enhancements that improve the performance of Pyrosequencing, especially in the areas of sequencing read length and first-time assay success.

What’s New

By adding Single-Stranded Binding Protein (SSB) to the enzyme mix in optimized quantities, sequencing read lengths have been greatly improved. SSB disrupts secondary structures in the template to be analysed, resulting in significantly longer read lengths. Other changes to the formulation result in increased peak heights, sharper peaks, and reduced background noise. All these factors add up to improve the accuracy and readability of Pyrograms.

Pyro Gold increases read length by decreasing drop-off, reducing background, and disrupting secondary structures. Sequence data from the 16S rRNA gene of Thiobacillus baregensis comparing the obtained sequence read length when using the old formulation (upper Pyrogram™) and the new Pyro Gold formulation (lower Pyrogram). In this case the read length has increased by a factor 3.

Longer Sequencing, Stronger Genotyping

For CpG methylation analysis, Pyro Gold can increase the number of CpG sites that can be quantified in a single assay.

Dramatically improved sequencing performance with Pyro Gold. This example shows the quantification of methylation in nine CpG sites spanning 82 nucleotides.

Improved First Time Assay Success

Another important overall effect of Pyro Gold chemistry is that Pyrosequencing is now even more ‘tolerant’ of challenging assay designs. In the set of 48 reference templates used to evaluate the performance of Pyro Gold, we included 13 templates that were previously difficult or impossible for Pyrosequencing to analyze. Out of these 13 templates, 12 were successfully analyzed using Pyro Gold without any changes to the assay.

In the 48 tested templates (which included 13 non-working assays), a significant increase in read length was demonstrated, and non-working assays were dramatically reduced.

Improved sequence read length and quality is important for short read sequencing for genetic and epigenetic analyses, and increases resolution and accuracy for sequencing applications such as microbial identification and drug resistance typing.

Pyro Gold products are designed for laboratory use only, which means they can be used either for research or by high complexity CLIA-certified labs.

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