Novel Strategies for Enhanced Detection of Hemoglobinopathy Carriers

SummaryThe overall goal of this project is to developvalidateand commercialize the first ever hemoglobinopathies panel kit on Oxford Nanopore TechnologiesNP systemcovering deletionsduplications and mutations in the alpha and beta globin genes associated with thalassemiasOur panel will detect phasing of silent carriers in alpha thalassemia and assess the risk for both alphaand beta thalassemiadespite a negative result from conventional methods of testingNo commercially available kit offers this range of capabilitiesThese tasks will accomplished by leveraging three enabling technologiesiAsuragen s proprietary AmplideXPCR chemistries for the amplification of complex DNA and DNA barcode implementationiiOxford Nanopore TechnologiesMinION systemthe first available NP platform for direct long read sequencing for the analysis of long and challenging stretches of DNAandiiiAsuragen s proprietary haplotype informatics phasing algorithmsStandard approaches for screening require a variety of cumbersome sequential tests that do not identify all carriersIn contrastour comprehensive test will assess carrier risk for alphaand beta thalassemiasascertain carrier status for alpha thalassemia with relevant in phase mutationsidentify the large disease causing deletions found in alpha and beta globin genesand provide clinically relevant copy number detection and phasing of the four alpha hemoglobin genesThe proposed test will further exploit this phasing approach to resolve sequence redundancy between the two genes that encode alpha hemoglobinHBAand HBAand accurately call andgtmutations within the HBB gene that causes beta thalassemiaComplementary wetware and software advancements will be integrated to overcome workflowplatformand assay related challenges and establish feasibility for the PCR NP phased sequencing approach for hemoglobinopathiesAsuragen s success in building end to end solutions for complex PCR and NGS assays by combining breakthrough chemistriessimple workflowsand sophisticated software suitesset a solid foundation for this project to develop a modularrd gen NGS solution for carrier screeningThe specific aims areAimEstablish a scalableand robust PCR based enrichment method for generating multiplexed andbarcoded HBAHBAand HBB gene cluster librariesthat is compatible with high throughput and highresolution phased sequencing on the ONT platformAimDevelop a novel NP sequencing analysis pipeline that enables comprehensive and automatedphasing of HBAand HBAand accurate detection of deletions and mutations of HBAHBAand HBBgene clusters known to cause hemoglobinopathiesAimDemonstrate feasibility for a unified single day workflow and analysis pipeline at aplex scale forthe MinION system and orplex for the Flongle systemusing gDNA from a clinically relevant set ofresidual blood specimens that span the range of hemoglobinopathy genotypes Narrative Hemoglobinopathies are genetic disorders that results in an abnormal structure of the hemoglobin moleculeand are among the most common inherited diseases worldwideaffectingpeople annuallyCarriers with genetic changes in the hemoglobin genes are present in about one inpeoplehoweverin locations under selection pressure due to endemic malariaas many asof individuals have variants associated with severe hemoglobinopathiesand people with these variants may have little to no symptoms and might not be identified by standard testingWe will develop a new carrier screening panel kit using cutting edge sequencing technology to address this unmet needs for cost effective testing