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11th Annual <strong>Sequencing</strong>, Finishing, and Analysis in the Future Meeting EXTENSIVE GENETIC HETEROGENEITY OF CIRCULATING RESPIRATORY SYNCYTIAL VIRUS STRAINS REVEALED BY TARGETED, NEXT GENERATION WHOLE GENOME RNA SEQUENCING Wednesday, 1st June 15:00 La Fonda Ballroom Talk (OS‐3.04) Darrell Dinwiddie 1 , Kurt Schwalm 1 , Walter Dehority 1 , Joshua Kennedy 2 , Stephen Gross 3 , Gary Schroth 3 , Stephen Young 4 1 University of New Mexico Health Sciences Center, 2 University of Arkansas for Medical Sciences Arkansas Children’s Hospital, 3 Illumina Inc, 4 TriCore Reference Laboratories Background: Respiratory syncytial virus (RSV) is a major cause of childhood morbidity. Significant annual fluctuations in incidence and severity occur, yet the specific genetic variation that influences transmission, virulence, and pathogenesis for RSV is poorly understood. Methods: We developed a hybridization‐based method to target and enrich complete coding sequences of respiratory syncytial virus from clinical samples. The enriched samples undergo deep sequencing in a high‐throughput, multiplexed, rapid manner on the Illumina MiSeq. A custom bioinformatic pipeline is used to determine specific viruses, construct nearly complete genome sequences, assess viral gene expression, detect genetic variation and conduct phylogenetic analysis. Results: We have completed deep, next generation sequencing of 102 RSV positive samples across four infection seasons. Of the samples sequenced, we have generated complete or nearly complete genomes from 69 of 102 (67.6%) samples and have covered >50% of the genome from 29 of 102 samples (28.4%). Together, 96% of samples sequenced have >50% of their genomes sequenced. Alignment of sequencing reads to their appropriate prototype RSV A (NC_001803) and RSV B (AY353550) reference sequences produced an average of 410 variants (range 257‐474) and 466 variants (range 418‐510), respectively. The average number of non‐synonymous variants was 79 for both RSV A and RSV B samples. Strain and phylogenetic analysis reveal the presence of at least 6 distinct strains of RSV co‐circulating during the same infection season. Conclusions: Taken together, our data reveal that current clinical RSV isolates differ from signifi‐ cantly for their reference sequences and suggest that genetic diversity of co‐circulating RSV strains during the same infection season may be underappreciated. Evaluation of RSV by targeted, deep, next generation RNA sequencing provides important information about clinical viral isolates cur‐ rently not detected by clinical testing that may reveal genetic factors that impact clinical severity of illness and inform clinical management. 21
11th Annual <strong>Sequencing</strong>, Finishing, and Analysis in the Future Meeting IMPLEMENTATION OF SEQUENCING PLATFORM IN GABON: NEEDS AND CHALLENGES FOR AN EFFICIENT EPIDEMIOLOGICAL SURVEILLANCE STRATEGY Wednesday, 1st June 15:20 La Fonda Ballroom Talk (OS‐3.05) Nicolas Berthet 1 , Ingrid Labouba 1 , Andy Nkili Meyong 1 , Patrick Chain 2 , Momchilo Vuyisich 2 , Tracy Erkkila 2 , Eric Leroy 1 1 Centre international de Recherches médicales de Franceville, 2 Los Alamos National Laboratory With the emergences of different viral pathogens in Africa such as Zika virus in Gabon in 2007 – 2010 (Grard et al:, PLoS Negl Trop Dis. 2014), Bas‐congo rhabdovirus in Central Africa in 2012 (Grard et al:, PLoS Pathog. 2012), or more recently Ebolavirus in West Africa in 2014, the pathogen discovery field should become a priority in biology research for concerned countries as well as for entire African continent. There, providing required tools for detection, identification and characterization of potential emergent infectious pathogens becomes primordial to diagnose first, then adequately manage outbreaks and finally implement appropriate epidemiological surveillance. The Centre International de Recherches Médicales de Franceville (CIRMF) located in south of Gabon focuses its research activities on human and animal infectious diseases. The main CIRMF’s objectives are the epidemiological surveillance of human and/or zoonotic viral outbreaks mainly in Central Africa. This naturally includes not only diagnosis activities for adequate follow‐up of population and neighbourhood; but also the identification of involved viral pathogens. CIRMF’s equipment for specific and generic analyses based on PCR technologies has made it an efficient infectious diagnosis centre for the sub‐region. Moreover, thanks to its installation of biosafety level 2, 3 and 4 the CIRMF hosts all required structures to support the treatment of high risk biological samples as well as different steps of in vitro pathogen isolation process. Finally the CIRMF was progressively equipped to answer to these public health needs. Basically the CIRMF was locally able to detect and identify viral pathogens. However, further characterization by sequencing was only possible via subcontracting with European laboratories. This represented a considerable limit in terms of delay especially during outbreaks when time is so precious. In absolute, to locally treat samples from reception on site to sequencing data analyses would constitute an important gain of time and would improve epidemiological surveillance of all Central African sub‐region. Since November 2013, thanks to DTRA financial and LANL technical supports the CIRMF worked for installing and optimizing a sequencing platform to complete its research ability in pathogen discovery field. We suggest here to present the challenges met by CIRMF et al. to implement this platform from the purchase of the sequencer to date while it is workable even if further improvement are still required. 22
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Reliable solutions for focused NGS
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