Genetic Engineering & Biotechnology News

AUG 2017

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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18 | AUGUST 2017 | | Genetic Engineering & Biotechnology News Kathleen Shelton and Beth Walczak, Ph.D. Understanding cellular diversity and dynam- ics in tumor microenvironments are critical for elucidating mechanisms of cancer pro- gression, response to therapeutics, and patient prognosis. Until recently, tumor analysis had primarily been accomplished by either ana- lyzing protein cell-surface markers via flow cytometry (a process that is more straightfor- ward for liquid tumors than for solid tumors), or by bulk RNA expression analysis of a het- erogeneous population of tumor cells. Neither solution provides a complete understanding of the mechanism of disease progression. For more than 40 years, BD has provided tools and analysis solutions for single-cell pro- tein expression analysis via Fluorescence Acti- vated Cell Sorting (FACS™). BD has recently added a suite of tools to address critical chal- lenges for single-cell gene-expression analysis: the BD single-cell gene-expression platform for targeted gene-expression profiling, and BD TuDoR™, a reagent for tissue dissociation. 1 These products create a standardized method for generating integrated protein and gene expression data at the single-cell level. Together, they provide a greater understand- ing of the tumor microenvironment and het- erogeneity within cell populations. The BD single-cell gene-expression plat- form is a complete system of reagents, instru- ments, and analysis software that works to- gether to provide digital, quantitative gene-ex- pression profiling of thousands of single cells via next-generation sequencing (NGS). The methodology enables the identification and profiling of rare cell types within a highly het- erogeneous population. Components include a novel planar array of 200,000 microwells for cell capture (single-cell cartridge), bead- based 3' RNA-seq assays, sample-loading sta- tion, cartridge scanner for QC analysis, and a suite of data analysis and visualization tools. BD's patented molecular-indexing tech- nology provides the foundation for the BD single-cell gene-expression assay. 2 This tech- nology captures mRNA molecules and im- mediately tags them with a cell-specific iden- tifier (cell barcode), plus a unique molecular index (UMI) for each individual mRNA. This methodology allows for the pooling of tens of thousands of cells into one tube for a BD Genomics' Tools Measure Cellular Diversity within a Tumor Culling Information from the Tumor Microenvironment OMICS Assay Tutorial Insights Genomics & Proteomics Next-generation sequencing (NGS) technology has been a boon for both researchers and technology development companies alike. In a small number of years, NGS has begun to efficiently transition from being purely a research tool to a tool that is used in the clinical arena. While it is still many steps away from replacing traditional gold-standard diagnostics, NGS technology has a promising future. One recent example lies with Helix, a personal genomics company, which operates one of the world's largest NGS laboratories in San Diego. The company recently announced that its laboratory was accredited by the College of American Pathologists (CAP) for meeting the highest standards in quality, operations, and patient safety. The CAP Accreditation, and the Clinical Laboratory Improvement Amendment (CLIA) certification, which Helix earned in 2015, are interna- tionally recognized credentials awarded to laboratories complying with the most comprehensive, rigorous, and scientifically endorsed standards of laboratory practice. "Our state-of-the-art, high-throughput DNA se- quencing laboratory is the foundation of our business and critical to the success of our soon-to-launch and first-of-its-kind online marketplace for DNA-powered insights," explained Robin Thurston, CEO at Helix. "We're proud to earn the dual distinction of being CLIA-certi- fied and CAP-accredited and commend our scientific and clinical teams for their diligence and commitment to uphold the industry's most stringent requirements for laboratory testing, record-keeping, reporting, safety, and quality." Helix utilizes a proprietary sequencing assay known as Exome+. The assay sequences more than 22,000 protein-coding genes—known as the exome—plus hundreds of thousands of additional information-rich regions. Additionally, the company is planning to launch the first online marketplace for DNA-powered products, enabling consumers to sequence their DNA to access personalized insights on health, fitness, nutrition, ancestry, family, and entertainment from its network of partners. n Sequencing Lab Receives Coveted CAP Accreditation Figure 1. The BD single-cell gene-expression assay captures 3' poly A mRNA molecules by using functionalized beads. Each bead contains a universal linker, a cell label (CL), a molecular index (MI), and an oligo dT. After cDNA is made through reverse transcription, the cells are pooled into one tube for targeted RNA library preparation. During library preparation, the required sequencing adapters are added. Figure 3. The graph compares the percentage of cancer stem cells detected in the primary tumor samples vs. metastatic lymph node samples using the BD single-cell Onco-BC panel. Figure 2. The plots are t-Stochastic Neighbor Embedding (t-SNE) projections of 24,952 single cells derived from disassociated primary and metastatic breast tumors, annotated by originating sample (A) or cell type (B). Figure 4. The graph in (A) compares sequencing reads per cell required for targeted RNA-seq vs. whole transcriptome RNA-seq (WTA), and (B) compares the number of molecular indices detected per 1,000 reads for targeted RNA-seq vs. whole transcriptome RNA-seq. A B

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