CHALLENGE:

Abstract

Using the design strategy highlighted above, we created a multiplexed AmpliSeqTM RNA primer pool that would provide receptor typing information as well as mutational analysis capabilities of the VH region of the B cell receptor. We received 4 B-CLL RNA samples from our customer and successfully created targeted RNA sequence libraries as shown by the Agilent2100 Bioanalyzer traces above. Although the yield was low for sample B, C, and D, we were able to get viable libraries in sufficient quantity for template prep and sequencing on the Ion Torrent PGM 318TM Chip. We also identified a unique data analysis tool that proved incredibly helpful in evaluating the resulting amplicon information. This analysis tool, IgBLAST (http://www.ncbi.nlm.nih.gov/igblast/) was developed specifically to map sequencing reads to the VH, D, and J domains of the B cell receptor and also display the variants identified in reads mapping to the VH region (6). Using this tool on 1000 reads of each of the 4 RNA libraries we were able to see significant alignments to the reference available with the online IgBLAST tool. For sample A-300903, we also identified the predominant B Cell Receptor form. Historically, the fluorescent in situ hybridization (FISH) technique has been widely employed to study genomic variation in malignant cells. Recently the field of molecular cytogenetics is investigating next generation sequencing technologies to get even more complete information about the genomic variation in malignancies (1). Recently a gene fusion between echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) was identified in a NSCLC patient with a smoking history leading to a phase I clinical trial with an inhibitor of ALK (2,3). Subsequently, additional genes have been identified to be involved in lung cancer and targeted for potential for therapeutic intervention, such as RET and ROS1 (4). The FISH technique is labor intensive and experimentally demanding limiting the number of individual fusion genes that can be reasonably assayed for in any given sample. Due to the inherent limitations of FISH and the increased identification of chimeric fusion kinases with potential for oncogenic transformation the desire was to find a technology that would allow for testing for multiple fusion genes with limited tissue. With the new advancements in targeted RNA sequencing, we attempted to design a single panel targeting over 40 fusion genes involving ALK, RET and ROS1. Testing tissue samples for these fusion genes can be accomplished in one amplification reaction from as little as 5 ng of FFPE RNA. ABSTRACT Life Technologies recently released the first Ion AmpliSeqTM offering for RNA which included a library construction kit as well as an integrated design system on Ampliseq.com. This platform allows researchers to simultaneously interrogate gene expression of up to 1200 targets in a single tube, thus combining the sensitivity of qPCR with sequence information gained from RNA sequencing. Since this launch, we have developed a program referred to as white glove to push the limits of what AmpliSeqTM RNA currently offers. Here, we describe several examples of how these white glove initiatives have resulted in technological advances which will expand the repertoire of targeted RNA sequencing applications. We adapted our Ion RNA AmpliSeqTM primer designer to enable design to multiple RNA transcript configurations addressing several biological phenomena. In particular, we have successfully designed and tested panels to investigate gene fusion detection in cancer, characterize B-cell receptor IgH stereotypes/mutational burden, and interrogate both cancer fusions as well as hotspot mutations in a single panel. In addition, we have developed software to design AmpliSeqTM RNA fusion detection assays given COSMIC annotation. To facilitate easy reuse of AmpliSeqTM RNA fusion primer designs, we created a database to store designs as they are created. Currently, this database contains over 400 fusion detection designs.