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ESHG14 Poster: NGS to Sanger Sequencing, Synchronizing Variant Files

442 views · Published 1 June 2014 · 1:40 · Indexed 26 October 2026

Channel: Thermo Fisher Scientific · 2014 · Science & Technology

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Download this poster: http://www.slideshare.net/Life-Technologies-Corp/eshg14-poster-p1670-m-synchronizing-ngs-and-ce-variant-files

See all our posters from ESHG: http://ioncommunity.lifetechnologies.com/community/behindthebench/blog/2014/06/01/eshg14-download-our-posters-here-save-on-travel-expense

Whole exome or panel sequencing projects performed by next generation sequencing (NGS) technologies typically reveal a large number of variants which may require verification by an orthogonal method. To that end, automated fluorescent Sanger sequencing is the method of choice since it is accurate, affordable and easy to perform. To facilitate the re-sequencing of any exon in the human genome we have recently made available to the scientific community a free to use tool called Primer Designer™ . The tool provides the designs for over 350.000 PCR primer pairs that cover 99% of all exons in the human genome. Amplicons generated with these primers can be readily sequenced using the BigDye®Direct, BigDye® Terminator v1.1 and v3.1 sequencing chemistries on the 3500xl Genetic Analyzer capillary electrophoresis platform. For variant identification the sequencing trace files are analyzed with Applied Biosystems Variant Reporter® Software which requires the import of a text file with a reference sequence for sequence alignment and comparison. Here we show the utility and workflow of a new on-line tool called VR Toolkit™ that generates a reference file from Primer Designer --derived PCR amplicons that contains the coordinates of the chromosomal location. Use of this annotated reference file in Variant Reporter® Software allows the generation of an output file that can be compared and matched to a variant call file (vcf) from an NGS instrument. The VR Toolkit enables the connection and synchronization between NGS data and traditional Sanger sequencing data analyzed with Variant Reporter® Software and should be of benefit for all researchers seeking to validate NGS data by Sanger sequencing.

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