Poster abstracts 67 phases. The first phase is to classify the cDNAs and the second is to complete fulllength sequencing and functional annotations. We have developed two original methods to construct full-length cDNAs efficiently: 'cap-trapper', which preferentially recognizes the Cap site of mRNA; and the 'trehalose-thermoactivated reverse transcriptase', which allows the reverse transcriptase reaction at higher (60 C) temperatures. We have constructed over 80 libraries from embryonic tissues of different developmental stages and adult tissues to ensure the greatest possible coverage of the expressed mRNA. More than 200,000 successful sequencing passes have been performed with the use of two tools developed in-house: a high-throughput plasmid preparation system and the RISA 384 capillary sequencer. Most of the sequences were performed from the 3´ end to select individual cDNAs. We have selected more than 30,000 different cDNAs. Using these sets of RIKEN full-length cDNA, we have established gene expression microarrays containing a 20 K set of RIKEN full-length cDNA unique mouse genes (http://genome.rtc.riken.go.jp). This set has been used to profile expression patterns of various adult and embryonic tissues. Target DNAs were PCR amplified and printed on Poly-L-lysine coated glass slides. Target DNAs were blocked by excess amounts of Cot1DNA. Probes were labelled by two-colour fluorescent dye using random primer and reverse transcriptase. Normalization has been achieved using a global normalization method. We have also developed a program to filter the noise. The experiment was done twice and reproducible results were extracted and clustered. We will present a large set of data that show the spatial and temporal expression patterns of mice. These mouse full-length 20 K cDNA microarrays are widely applicable to analyse the global expression profiling of normal and diseased status of mice.
Bacterial alkaline phosphatases (APases), except those isolated from Bacillus licheniformis, are approximately 45-kDa proteins while eucaryotic alkaline phosphatases are 60 kDa. To answer the question of whether the apparent 60-kDa alkaline phosphatase from Bacillus licheniformis accurately reflected the size of the protein, the entire gene was analyzed. DNA sequence analysis of the alkaline phosphatase I (APaseI) gene of B. licheniformis MC14 indicated that the gene could code for a 60-kDa protein of 553 amino acids. The deduced protein sequence of APaseI showed about 32% identity to those of B. subtilis APase III and IV and had apparent sequence homologies in the core structure and active sites that are conserved among APases of various sources. The extra carboxy-terminal sequence of APaseI, which made the enzyme bigger than other procaryotic APases, was not homologous to those of eucaryotic APases. The amino acid composition of APaseI was most similar to that of salt-dependent APase among the isozymes of B. licheniformis MC14. Another open reading frame of 261 amino acids was present 142 nucleotide upstream of the APaseI gene and its predicted amino acid sequence showed 68% identity to that of glucose dehydrogenase of B. megaterium.
Introduction Detection of oncogenic fusions has been of great importance for understanding tumorigenesis and for precision oncology in enhancing diagnosis and selection of targeted therapies. Herein, we describe an extended Oncomine targeted RNA sequencing assay for detection of fusion transcripts and intragenic rearrangements (exon deletion/skipping). For multiple key driver genes we also supplemented the panel with a complementary transcript-based expression imbalance assay designed to identify gene fusions in a partner agnostic manner. Methods Based on evidence from Oncomine™ Knowledgebase and collaboration with leading oncology researchers, we designed an Ion AmpliSeq™ panel to target > 1,200 fusion breakpoints in > 50 driver genes, > 40 intragenic rearrangements (e.g., MET exon 14 skipping, ARv7, EGFRvIII) in 7 genes, and 5 RNA expression controls. In addition, the panel supports detection and reporting of non-targeted fusions (i.e., novel combinations of drivers and partners). We supplemented the panel with exon tiling expression imbalance assays, using amplicons tiling the exon junctions of ALK, RET, NTRK1, NTRK2 and NTRK3 to measure 3'/5' expression imbalance signatures. We developed a bioinformatic tool to call fusions from a normalized and corrected expression imbalance profile per gene (using a baseline from normal formaldehyde fixed paraffin embedded [FFPE samples]). We optimized the gene fusion algorithms and integrated them as workflows into the Ion ReporterTM Software to facilitate the summary of the results with relevant annotations, rich data visualizations and easily interpretable reports. Results We sequenced hundreds of positive and negative fusion samples including commercial reference standards, cell lines and FFPE clinical research samples on the Ion GeneStudioTM S5 sequencer. To assess the feasibility of the combined panel, we sequenced the Seraseq® FFPE tumor fusion RNA reference, 7 fusion positive cell lines with ALK, RET, ROS1, NTRK1, FGFR1, FGFR2 and FGFR3 rearrangements, and cohorts of FFPE samples using 20ng RNA as input and successfully detected the expected fusion isoforms or other RNA rearrangements in each sample. We applied the exon tiling fusion detection method for ALK, RET and NTRK1 and observed perfect concordance between the true isoform in the positive samples and the predicted breakpoint position and magnitude of 3'/5' expression imbalance indicated by the exon tiling method. Conclusions We developed an extended, multiplexed RNA panel for fusions and intragenic rearrangements that retains the simple workflow and fast turn-around time of previous Oncomine fusion panels and significantly expands the scope of fusion isoform detection including methods to detect gene fusions in a partner agnostic manner. For research use only. Not for use in diagnostic procedures. Citation Format: Amir Marcovitz, Rajesh K. Gottimukkala, Gary G. Bee, Jennifer M. Kilzer, Vinay K. Mital, Elain Wong-Ho, Chenchen Yang, Yu-Ting Tseng, Scott P. Myrand, Paul D. Williams, Seth Sadis, Fiona C. Hyland. RNA sequencing based gene fusion detection with oncomine comprehensive assay plus [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 177.
Comprehensive genomic profiling (CGP) of tumor samples by next-generation sequencing is used to support clinical and translational research into the genetic variants that serve as biomarkers for diagnosis, prognosis and potential therapeutic response. However, as these assays grow in size to meet the expanding demands of users, it is challenging to maintain performance in the face of limited sample input necessitated by small sample volumes and to provide a simple and fast sample to report workflow with limited hands-on time. We, therefore, developed OCA Plus to meet user needs for a large CGP assay with excellent performance. Gene content was prioritized based on potential clinical relevance and variant prevalence in solid tumors. Over 500 genes were selected including genes in the indication statements of approved drug labels, clinical guidelines, and in the enrollment criteria of clinical trials. In addition, driver genes were selected in key pathways including DNA repair and immune checkpoint response. Amplicon design strategies were optimized accordingly for key hotspots, full coding sequences or copy number variation (CNV). The assay used Ion AmpliSeq™ technology with manual library preparation or automated templating on the Ion Chef™ System and sequencing on the Ion GeneStudio™ S5 platform. Twenty ng of purified DNA was routinely used as input. An automated tumor-only workflow for variant calling and sample quality reporting was provided within Ion Reporter™ Software. Streamlined access to reporting of variant relevance was enabled by Oncomine™ Reporter. In development studies of cancer cell line and formaldehyde-fixed, paraffin-embedded (FFPE) tumor samples, the assay displayed excellent uniformity (98% and 94%, respectively). Detection of single nucleotide variants and indels in cell lines and FFPE samples showed >95% sensitivity and PPV. Detection of CNV gain and loss in cell lines and FFPE samples showed >95% sensitivity and PPV. Assessment of tumor mutational burden (TMB) using publicly available whole-exome cancer sequencing data as well as test cell lines and FFPE samples showed high concordance with whole exome sequencing (R2 > 0.90). MSI sensitivity and specificity was >95% as tested using a diverse set of tumor samples. Targeted fusions were reported with 100% sensitivity and specificity when tested with commercially available controls. Total time from purified DNA to end of sequencing was < 2 days with < 3 hours of hands-on time and the time from post-sequencing to report generation was < 2 hours. Oncomine Comprehensive Assay (OCA Plus) was developed to support CGP and routine clinical research in oncology. The assay design and informatics workflow were optimized to support low input and rapid sample-to-report turn-around time, which will accelerate clinical and translational research. Citation Format: Vinay Mittal, Jennifer Kilzer, Dinesh Cyanam, Janice Au-Young, Santhoshi Bandla, Gary Bee, Sameh El-Difrawy, Aren Ewing, Rajesh Gottimukkala, Mohit Gupta, Nickolay Khazanov, Anelia Kraltcheva, Amir Marcovitz, Scott Myrand, Rose Putler, Yu-Ting Tseng, Warren Tom, Cristina Van Loy, James Veitch, Paul Williams, Elaine Wong-Ho, Huimin Xie, Chenchen Yang, Zheng Zang, Seth Sadis. Comprehensive genomic profiling of solid tumors for key targeted and immuno-oncology biomarkers using Ion Torrent NGS technology on the Oncomine Comprehensive Assay Plus (OCA Plus) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 179.
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