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UK Researchers Update PCR Primer Search Tool with Data from 1000 Genomes Project


Developers at the National Genetics Reference Laboratory in Manchester, UK, have released a new version of SNPCheck, a web-based tool that lets scientists check for common SNPs in oligonucleotide primers or probes that could reduce annealing efficiency in PCR experiments and ultimately lead to false results.

This release includes an option for users to check primers or probes against data from the 1000 Genomes Project. Previously, SNPCheck users only had access to data from the National Center for Biotechnology Information’s Single Nucleotide Polymorphism database.

SNPCheck was developed for use in diagnostic laboratories, where high levels of quality assurance are an essential part of primer and probe design. But it can be used by any academic research groups and commercial companies that use human genome sequence data in their studies and projects, Andrew Devereau, NGRL’s director and a co-developer of SNPCheck, told BioInform.

Specifically, the tool helps users avoid “allelic dropout” — a situation where an allele isn’t amplified efficiently during PCR and is underrepresented in the post-PCR sample. This could lead to false positive or false negative results in genetic testing. Allelic dropout is often caused by the presence of SNPs in the section of DNA that has been chosen as a primer binding site.

SNPCheck uses Blast to search for sequences that match a user’s primers and probes. Once it identifies the most likely binding sites, it then searches for SNPs in those sites against data from the latest build of dbSNP and now from the current 1000 Genomes Project data. The results are presented in a tabular summary along with diagrams for each pair of primers.

The system allows users to check single primer pairs for free. Those who want to check large numbers of primers simultaneously — up to 500 primer pairs, for example — can pay for an annual subscription that provides access to batch-checking functions as well as storage resources that let users save their SNPCheck results and primer sets in their laboratory accounts and check for new SNPs as new datasets become available.

An annual subscription costs around $274 (£170) per lab, a fee that covers access for multiple users, Jasmin Opitz, a health informatics specialist and one of SNPCheck’s developers, told BioInform.

NGRL adopted its subscription-based model this past June so that it could generate the funds needed to maintain SNPCheck in the long run, Devereau explained. The tool was initially developed with funding from the UK’s Department of Health.

So far, SNPCheck has around 111 subscribing labs with multiple users as well as several researchers who use the free version to check single primer or probe pairs, Opitz said.

She also said that the developers are working on incorporating new datasets into SNPCheck from recent sequencing projects to ensure that it includes the most up-to-date information for checking primers and probes.

The developers decided to include data from the 1000 Genomes Project because it offers more population frequency information and more uniform coverage than dbSNP, Devereau explained.

“We were aware that the dbSNP data wasn’t the best source of data” due to its heterogeneity,” he told BioInform. Furthermore, although there is some data from the 1000 Genomes Project in dbSNP, new database builds are released infrequently, which means that SNPCheck would only be updated when a new dataset comes out, he added.

By including data from 1000 Genomes directly, the developers will be able to incorporate new data as it is released from the project rather than waiting for updated versions of dbSNP, he said.

Moving forward, the developers intend to incorporate additional datasets so that they can provide “more detailed information [and] better and more comprehensive coverage,” Devereau said.

For their next steps, they plan to add data from the National Heart, Lung and BIood Exome Sequencing Project's exome variant server, Opitz said.

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