Development of a kit for the targeted depletion of abundant sequencesfromDNA/RNA libraries

ABSTRACT Next generation sequencingNGShas the potential to revolutionize how infectious disease is diagnosed and treatedenabling the detection of a wide variety of pathogens in a single testHoweverdespite the increasing adoption of molecular diagnostics in clinical microbiology labsNGS has not become widespread due to its costturnaround timeand the technical and computational expertise required to produce and analyze the dataTo overcome these challengesArc Bio is developing a suite of technologies that will enable clinical microbiology labs to implement NGS pipelinesThe Arc Bio Infectious Disease PipelineIDPproduct includes all the experimental protocols and software necessary to go from sample to answer for pathogen detection in clinical samplesincluding novel tools to make pathogen sequencing less expensivemore sensitiveand more accurateOne of these tools is a Casbased host DNA RNA depletion technology that was developed in Phase I of this projectDepletion of host sequences dramatically reduces the cost and increases sensitivity when sequencing primary samples such as plasma and cerebrospinal fluidWe have achieved the goals set forth in that proposalincludingdepletion of rRNA anddepletion of abundant transcriptome sequences from RNAseq librariesfurthermorewe demonstratefold enrichment of HIV sequences in depleted sampleswith no detectable off target depletionIn Phase IIwe will develop and validate our IDP toward sequencing samples from HIV infected and immunocompromised individualsThis will involve the following activitiesoptimizing and scaling up our host depletion technologycompleting development of our proprietary graph based analytics softwarewhich enables accurate strain level detection thatlearnsas more data is acquiredandvalidating our complete pipeline on samples from healthy individuals for baseliningas well as those from patients infected with HIVHCVand HBVBy performing unbiased next generation sequencing with host depletion on these sampleswe will be able to report not only viral genotype and drug resistance mutationsbut also viral titer and co infectionssuch as hepatitis B and CThe result of this project will be a completevalidated pipeline that will include all protocols and software necessary for initial deployment into alpha partner clinical labs Project Narrative Next generation sequencing of clinical samples for pathogen detection is likely to replace many existing targeted molecular diagnostic tests in the near futureimproving our ability to detect and treat infectious diseaseHoweverbefore this replacement can occurthe technology must become less expensivefasterand more user friendlyIn the proposed workArc Bio will develop and deploy a streamlined sample to answer pipeline that will use its host DNA RNA depletion technology to reduce sequencing costs and increase sensitivityand its proprietary bioinformatics software to enable rapidaccurateand scalable data analysis with a user friendly interface