Genetic Engineering & Biotechnology News

AUG 2016

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34 | AUGUST 2016 | GENengnews.com | Genetic Engineering & Biotechnology News The wet bench includes pre- and post-an- alytical checkpoints: DNA extraction, DNA quality, library preparation, and quantifica- tion. The dry bench includes bioinformatics steps: post-sequencing run, sample, and vari- ant metrics. For each run, the metrics verified are as follows: chip loading, usable sequences, polyclonality, and low-quality reads. Addi- tional values that may be assessed for each in- dividual sample include coverage uniformity and on-target reads. The complete NGS process can be moni- tored step by step. Dr. de Abreu explains that in addition to the mandatory routine testing of blinded CAP samples, many CLIA labora- tories also agree to participate in additional proficiency testing through inter-laboratory sample exchanges. For its part, the Dartmouth- Hitchcock Medical Center has organized a Next-Generation Sequencing Project Team. It reviews and updates the accreditation checklist requirements specific to NGS to adapt them to meet the rapid evolution of NGS and its trans- lation to clinical diagnostic testing. The Center's laboratory for Clinical Ge- nomics and Advanced Technology (CGAT) analyzed over 1,700 FFPE tumor tissues com- posed of multiple tumor types, with about 80% passing the "wet bench" QC check- points. A large percentage of identified somatic mutations were actionable. The Dartmouth-Hitchcock Medical Cen- ter established the Multidisciplinary Molecu- lar Tumor Board to evaluate potential treat- ments for the actionable cases identified by the sequencing laboratory. In over 50% cas- es, the board was able to recommend ther- apy with a targeted agent. In a few patients treated with the recommended therapy, dis- ease outcomes were positive, suggesting that increasing the awareness among patients and clinicians of the benefits of molecular testing could improve patient care. NGS Continued from page 33 TRANSLATIONAL MEDICINE > UMich Cancer Center to Use Trovagene ctDNA Urine and Blood Tests in Pancreatic Research Trovagene initiated a multiphased collaborative research program with the University of Michigan Compre- hensive Cancer Center utilizing the Tro- vera™ KRAS ctDNA liquid biopsy test. "KRAS gene mutations occur in over 90% of pancreatic carcinomas. There is an urgent need for targeted therapies and a precision diagnostic test to identify who would benefit from these therapies," said Diane Simeone, M.D., director of the Pancre- atic Cancer Center at the University of Michigan Comprehensive Cancer Center. "As part of this research col- laboration, Trovagene's ctDNA urine and blood tests will be utilized as noninvasive diagnostic tools to enable early detection and rapid monitoring of patient response to therapy." > Exosome Dx and Takeda Partner to Develop RNA-Seq Platform for Biomarkers Exosome Diagnostics will work with Takeda Pharmaceuticals to de- velop an exosomal RNA sequencing platform for biomarkers. As part of the agreement, Exosome Diagnos- tics will establish a gene-expression pipeline with Takeda that will utilize its exosomal RNA isolation technol- ogy, RNA-Seq biomarker discovery platform, proprietary algorithms, signal enhancement technology, and additional ancillary technologies for analysis of exosomal RNA. The goal of the pipeline is to develop a platform for serial analysis of gene expression patterns in patients. > Genomic Health to Commer- cialize Epic Sciences' Prostate Cancer Liquid Biopsy Test Genomic Health will commercial- ize Epic Sciences' AR-V7 liquid biopsy test in the U.S. The blood-based test detects the V7 variant of the andro- gen receptor protein (AR-V7) in the nucleus of circulating tumor cells (CTC)—information that can help guide treatment selection in patients with metastatic castration-resistant prostate cancer (mCRPC). Genomic Health will have exclu- sive distribution rights to market and sell the new Epic Sciences liquid biopsy test in the U.S. beginning in early 2017. As part of the agreement, Genomic Health will make an equity investment in Epic Sciences. n News Molecular Diagnostics Earlier this year, a team of scientists from Directed Genomics and New England Biolabs ® (NEB ® ) presented a poster entitled "Application of target enrichment combined with unique molecular identifiers to determine allelic frequencies of human cancers" at the AACR conference in New Orleans. The study fo- cused on the use of NEBNext Direct™, a target-en- richment technique developed for hybridization- based capture of genomic regions of interest. When this method is used, target genomic DNA sequences are isolated and converted into an Illumina-compatible library within seven hours. Unlike some alternative approaches that convert the entire genome into a library and select the targets as a final step, NEBNext Direct target-enrichment enzymatically removes off-tar- get sequences and converts only target regions of the native DNA molecules into sequencer-ready libraries, according to Andrew Barry, target enrich- ment product marketing manager, NEB. Target-enrichment and library-preparation strategies for next-generation sequencing typi- cally utilize PCR amplification steps that intro- duce substantial bias across amplicons. They can also lead to the creation of duplicate sequence reads, which in turn affects the quantification accuracy of somatic allele frequen- cies, an important factor in understanding tumor progression. Unique molecular identifiers (UMIs) are molecular tags that label each molecule prior to library amplifica- tion with a 12-basepair randomized sequence incorporated into the the library adaptor. The UMI, notes Barry, allows sequence reads to be disambiguated as PCR duplicates, enabling an accurate assessment of variant allele frequen- cies and molecular copy number of the origi- nal sample. "In this study, we enriched control samples of known variant allele frequencies representing chal- lenging sample types with the NEBNext Direct Can- cer HotSpot Panel, and we used the incorporated UMIs to determine allelic frequencies of select cancer targets," explains Barry. "This novel approach to target enrichment in conjunction with library preparation and the inclusion of UMIs enables filtering of PCR duplicate reads, offering improved sensitivity and more accurate assessment of variant allele frequencies." n Determining Allelic Frequencies of Human Cancers The NEBNext Direct Cancer Ho t S p o t Pa n e l d i s p l a y s high uniformity of coverage across targets. Target bases were sequenced to at least 50%, 33%, and 25% of the mean read depth. Several techniques have been em- ployed to determine copy number, including array comparative genomic hybridization (aCGH) and quantitative PCR (qPCR). However, none of these methods are amenable to high-throughput, directed copy number variation (CNV) detection, according to a research team from the City of Hope National Medical Center and Archer DX. In a poster on detecting CNVs in clini- cal samples, Haimes et al. described the development of a directed next- generation sequencing (NGS)-based method to rapidly and quantitatively measure the copy number of tens and potentially hundreds of genes simulta- neously. "This complete workflow, found in the Archer™ Universal DNA Kit, is powered by Anchored Multiplex PCR (AMP™) chemistry and processes dozens of samples in about six hours," explained Josh Stahl, CSO and general manager. "By ligating a molecular bar- code to randomly fragmented input DNA and then using AMP to simulta- neously enrich for several regions of each target gene, we can accurately measure the relative copy number of each target gene in test samples by counting unique molecular barcodes associated with each target region." The scientists validated their meth- odology with a 25-gene panel on a subset of NCI-60 cell lines by compar- ing their copy number measurements to those determined by both aCGH and qPCR. Results from both orthogo- nal methods strongly correlated with data from the team's NGS-based method. "We multiplexed hundreds of sam- ples on a single MiSeq® run and de- tected CNVs, both amplifications and deletions, of 2× magnitudes (and often lower) at extremely high confidence, indicating that this panel is amenable to highly multiplexed screens of potentially hundreds of samples," added Stahl. "Furthermore, we demonstrated that our NGS-based CNV detection workflow and analysis is compatible with DNA extracted from formalin- fixed, paraffin-embedded (FFPE) samples, suggesting that this system could be adapted for use in clinical applications." n Detecting CNVs in Clinical Samples Archer Universal DNA reagents are now integrated with the Archer VariantPlex system, which can generate target-specific libraries for next-generation sequencing.

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