Genetic Engineering & Biotechnology News

MAY15 2017

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Genetic Engineering & Biotechnology News | GENengnews.com | MAY 15, 2017 | 11 Bioinformatics Pipelines The bioinformatics pipeline (a set of ordered scripts that take raw data to data product to analysis results) is used to align reads from NGS assays, do variant calling, and filter mutations. "We download the ref- erence sequence from the National Center for Biotechnology Information (NCBI) web- site," informed Dr. Hegde. "And we design our own bioinformatics pipeline by writing scripts to pull regions we want to interro- gate clinically." The major step in pipeline validation is to check your assay sequence data, such as data from 5,000 known disease-causing genes in WES assays, against the reference sequence pulled. "There should be a one-to-one se- quence match if your assay was designed properly," asserted Dr. Hegde. When different pipelines are used for biomarker validation, results vary. "If a re- searcher uses five different pipelines for the same raw data, the overlapping mutation rate is only about 50%," Dr. Qiu pointed out. "If you're getting results from different laboratories, you cannot compare the data unless you do a unified analysis by the same pipeline." Whole Exomes for High Coverage Validation for genetic biomarkers must ensure that "you are not dropping out re- gions of clinical importance," insisted Dr. Hegde. "The key aspect of validation for ge- netic biomarkers is to make sure the assay design itself is accurate." Mutations in rare diseases could be any- where in the genome. There are 22,000 genes in the human genome and about 5,000 known disease-causing genes. WES screening allows interrogation of all 22,000 genes in one assay. "The 5,000 genes must be proper- ly annotated, not just to clarify what is hap- pening at the gene level, but also to know whether the genes may cause a disease—and to identify which downstream steps should be taken once the disease is identified," em- phasized Dr. Hegde. Dr. Hegde leverages NCBI's genome an- notation and also does a tremendous amount of bioinformatics validation work to identify errors and make sure gene information is ac- curately and appropriately coded. The WES assay design must include full, accurate cov- erage of the 5,000 disease-causing genes as well as inclusion of the 17,000 other genes. For WES assay validation, clinical labo- ratory improvement amendments (CLIA) re- quire establishment of a sample type, such as whole blood or saliva for DNA extraction. Dr. Hegde says they can also opt in already extracted DNA such as DNA NA12878, from NIST's Genome in a Bottle project, which has already been sequenced and facili- tates validation. Ultimately, the bioinformatics pipeline must be able to properly annotate sequenc- ing data obtained from clinical samples to help identify, discover, or rule out disease- causing biomarkers. Once annotation is completed, a board-certified clinical mo- lecular geneticist will determine whether sequence variants in the rare disease sample should be classified as benign, disease caus- ing, or indeterminable. EFIRM Electrifies Liquid Biopsy "Our analytical platform is called elec- tric field-induced release and measurement, or EFIRM," said Wong. "We are excited to bring it to liquid biopsy. EFIRM is an emerg- ing technology with performance advantages over current digital polymerase chain reac- tion (PCR) and NGS technology." EFIRM allows for rapid and direct detec- tion of nucleic acids, such as circulating tu- mor DNA mutations, in less than one drop of saliva, blood, or urine. Other liquid biopsy technologies require 10 mL blood or 100 mL urine. The National Cancer Institute recently funded a project for advancing EFIRM-liq- uid biopsy (eLB) to a CLIA-certified labora- tory developed test for detecting actionable EGRF mutations in patients with non-small cell lung cancer (NSCLC). EFIRM is a hybridization reaction that may be run in less than 15 minutes. Mutated sequences in the biofluid actively hybridize to an immobilized oligonucleotide capture probe sequence. Then, a horseradish per- oxidase reporter/detector probe containing a complementary sequence also hybridizes to the captured mutated sequence, which is detected through a substrate reaction. "The small volume and electric field allow a very specific reaction and reporting of the mutant sequence," Wong explained. The electric field pulsates rapidly, lysing exosomes present in the biofluid and re- leasing their molecular contents, including DNA, RNA, and protein, into the biofluid. Target tumor DNA trapped within the exo- somes is freed and can quickly be captured by the capture probe sequence. "A proof-of-concept clinical validation in two small, published pilot studies showed eLB detected actionable EGFR mutations in patients' saliva with 95% concordance with genotyped biopsies," asserted Wong. Com- prehensive EFIRM assay validation studies will be done with plasma from 300 geno- typed cases of NSCLC. A reference labora- tory will do an independent evaluation on the same plasma with digital PCR. "Thus, we will have a head-to-head comparison be- tween EFIRM, genotyped samples, and digi- tal PCR," concluded Wong. Drug Discovery U N S T O P P A B L E P U L S E 100x more sensitivity, down to the femtogram. 20x faster: separate up to 200,000 base pairs in about 1 hour. SHATTER ALL EXPECTATIONS IN GENOMIC DISCOVERY. Automating genomic discover y A AT I - U S . C O M

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