The aim of this proposal is to develop a bioinformatics pipeline, or Integrated Bioinformatics Analysis Suite (IBAS), specific for ultra-deep, targeted amplicon sequencing on the new Ion Torrent Personal Genome Machine (PGM). This will improve the accuracy of variation detection and enable analysis of genetic variations in different types of cancer. The technology used in the PGM is based on non-optical detection with semiconductor technology and so is sufficiently unique to enable its use as an orthogonal platform for confirmatory testing when other technologies are used for initial sequencing. The PGM offers a significant advantage over other methods of next generation sequencing by enabling the process of sample preparation to data generation to be completed in one day. However, it suffers from the inherent limitations of the technology, mainly errors at the 32 end. Additionally, currently available SNP callers are not particularly suited to high-throughput, ultra deep amplicon sequencing and there are few algorithms available for detecting other types of genomic variations. The proposed pipeline aims to address these issues and fill the urgent need for bioinformatics optimized for the panel-designed amplicon sequencing technology employed in the PGM. To achieve the proposed aims, 1) an IBAS that incorporates different approaches to read preprocessing, alignment to reference sequences, and error modeling to enable detection of single nucleotide polymorphisms (SNPS), insertion/deletions (indels), copy number variation (CNV) and other structural variation will be constructed;and 2) a comprehensive evaluation of the sources of variation will be performed, and the IBAS optimized for amplicon sequencing. The optimized pipeline will then be validated by processing data from the PGM for orthogonal validation of mutations detected in clinical thyroid fine needle aspiration biopsies. Successful outcome will enable Asuragen to provide a high-throughput ultra-deep sequencing service for clinical research and diagnostics.

Public Health Relevance

We aim to develop a bioinformatics application specific for the technology employed in the recently launched Ion Torrent Personal Genome Machine (PGM) that will improve the accuracy of detecting genetic variations in different types of cancer. The PGM is a compact, bench-top next generation sequencing instrument that enables rapid sequencing of an entire genome and at a much lower cost than established systems. The application will be used by Asuragen to offer a high-throughput sequencing service for clinical research and diagnostics.

National Institute of Health (NIH)
National Human Genome Research Institute (NHGRI)
Small Business Innovation Research Grants (SBIR) - Phase I (R43)
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Special Emphasis Panel (ZRG1-IMST-K (14))
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Bonazzi, Vivien
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Asuragen, Inc.
United States
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