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RNA-Based Gene Fusion Detection: Next Generation Sequencing Applications and Sample Workflow Improvements

Josh Stahl is the Chief Scientific Officer and General Manager for ArcherDx, Inc., where he has worked since 2013.  Mr. Stahl holds a Master’s in Biochemistry and an MBA from the University of Colorado at Boulder.  He joined ArcherDx in the company’s infancy, specifically to be part of something that would make a lasting impact on healthcare.  During his time at ArcherDx, the company has continuously developed new, innovative methods for DNA and RNA variant detection through next generation sequencing (NGS) for translational and oncology researchers.
  • Joshua Stahl, M.Sc., ArcherDx, Inc.

  • Chief Scientific Officer

  • Original Webinar Date: Tuesday, April 26, 2016

Join us for this co-sponsored webinar with ArcherDx, Inc.

This webinar will detail the advantages of gene fusion detection via RNA-Seq methods and enhancements that can be gained through improvements in nucleic acid purification protocols.

Gene fusions have been implicated as oncogenic drivers in an array of human cancers and arise from genomic aberrations such as translocations, insertions and deletions (indels) and inversions. Gene fusions can promote tumorigenesis by induced oncogene expression due to promoter insertion or by production of chimeric proteins with altered functionality. Tumorigenesis driven by kinase gene fusions can be blocked by kinase inhibitors, highlighting the importance of detecting these gene fusions in clinical samples.

Conventionally, fluorescence in situ hybridization (FISH) is used to detect gene fusions in clinical samples. As FISH alone cannot identify fusion breakpoints and unknown fusion partners, it is often coupled with RT-PCR and Sanger sequencing to detect novel fusions. However, both FISH and RT-PCR lack scalability, as only a few genes can be interrogated simultaneously. As gene fusions often induce expression, immunohistochemistry (IHC) can be used to infer the presence of a gene fusion based on protein expression level. Still, IHC lacks scalability, can’t identify breakpoints and misses fusions that do not alter gene expression. As FISH and IHC are rather subjective, breakpoint identification by direct sequencing is a superior tool to detect expressed gene fusions.

The advent of next-generation sequencing (NGS) technologies has enabled the rapid identification of unknown fusion genes. To detect gene fusions by NGS, genomic DNA can be used for whole genome or whole exome sequencing, or RNA can be used for whole transcriptome sequencing (RNA-Seq). In comparison to whole genome sequencing, which interrogates large intronic segments that likely contain biologically irrelevant fusions, whole exome sequencing only assays the exons. However, the additional exon enrichment step (hybrid capture) involved in whole exome sequencing adds to the overall cost and time to perform these assays. RNA-Seq, which interrogates only expressed fusions, is a faster, more cost-effective approach to detect biologically relevant gene fusions. Though RNA-Seq is somewhat limited in its ability to detect low expression levels of transcripts, target-enrichment strategies can be used to increase read depth over target regions of interest, thereby enhancing detection sensitivity.

Anchored Multiplex PCR (AMP) is one such target-enrichment strategy that significantly increases the sensitivity of gene fusion detection by RNA-Seq, enabling detection of chimeric transcripts with single-molecule resolution.  In contrast to amplicon-based target-enrichments methods, which require prior knowledge of fusion partners for opposing primer design, AMP uses unidirectional gene-specific primers and molecular barcode adaptors ligated to random start sites to enrich for both known and unknown fusions. With AMP-based RNA-Seq, a 100kb intronic sequence can be covered with one primer and 200,000 reads. Therefore, AMP-based RNA-Seq detects novel, expressed fusion genes with high sensitivity.

Nucleic acid extraction from clinical specimens, typically formalin-fixed paraffin-embedded (FFPE) samples, required for NGS assays remains challenging. Formalin-induced nucleic acid modifications, sample age and storage conditions result in low yields of poor quality nucleic acids. Target-enrichment by AMP helps overcome this issue, since as little as 20ng input RNA from FFPE can be used successfully. Methods to extract nucleic acids from FFPE samples are not standardized and are not automated, therefore lacking the scalability required in high-throughput sequencing labs. This webinar discusses the advantages of AMP-based RNA-Seq for fusion detection and the enhancement of this approach by using an automated total nucleic acid purification method, resulting in longer reads and higher complexity libraries.