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11th Annual Sequencing, Finishing, and Analysis in the Future Meeting EXTENDED VIRAL EXPLORATION OF HEMORRHAGIC FEVER SYNDROMES WITH NEGATIVE EBOLA DIAGNOSIS Wednesday, 1st June 20:00 La Fonda Mezzanine (2nd Floor) Poster (PS‐2b.01) Ingrid Labouba 1 , Andy Nkili Meyong 1 , Patrick Chain 2 , Maganga Gael 1 , Momchilo Vuyisich 2 , Tracy Erkkila 2 , Eric Leroy 1 , Nicolas Berthet 1 1 Centre international de recherches médicales de Franceville (CIRMF GABON), 2 Los Alamos National Laboratory Background: While in West Africa the hugest Ebola virus disease (EVD) outbreak was in progress, on 26 August 2014 the WHO reported another one in the North‐West of the Democratic Republic of Congo (DRC), in the district of Bouende (Equateur province) located at 1200km of North of Kinshasa. Since the virus was discovered in 1976, this was the 7th in this country. On 7 October 2014, a total of 69 cases (3 suspected, 28 probable, 38 confirmed) and 49 deaths (21 males, 28 females) have been recorded. 33 Blood samples collected from those patients were sent to the CIRMF for diagnosis confirmation and complementary investigation. Based on qPCR specific diagnosis, 7/33 cases were found Ebola Zaire positive (Maganga et al., NJEM 2014). 26/33 remaining samples were found negative for Ebolaviruses (Zaire, Bundibyo, Sudan species) and Marburg virus specific diagnosis as well as for generic Pan Filoviruses PCR. These then constitute a cohort of cases with an unknown etiology that require a deeper exploration. Here we suggest to investigate the presence of potential infectious causal agent able to be associated with hemorrhagic fever syndrome (HFS) in those patients found negative for any filoviruses. Methods: Filovirus‐negative whole blood samples were stored at ‐80°C until their total RNA extraction initiated under high security conditions in BSL‐4 and completed in BSL‐3 laboratories. After a ribosomal RNA removal, remaining RNA will be used for preparing DNA libraries directly and after a whole genome amplification (WTA) step to increase chances of viral pathogen detection by high throughput sequencing. In parallel, we will proceed to the isolation of potential pathogens by cell culture or mouse brain inoculation. Positive generated isolate or inoculate will also be treated for high throughput sequencing. Subsequent analyses will be performed by bioinformatician using CLC and EDGE softwares. Expected results and perspectives: Here we aim to identify the other potential infectious causal agents, different from Ebola and Marburg viruses that stroke during 2014 DRC outbreak. This project also aims to implement a standardized procedure for an extended and complete exploration of clinical syndromes with unknown etiologies. Currently dedicated to this small cohort of HFS patients, it will be used in the long term on entire CIRMF’s biobank and more. Funding Sources: Defense Threat Reduction Agency Agence Nationale de la Recherche FRANCE 89
11th Annual Sequencing, Finishing, and Analysis in the Future Meeting EVALUATION AND OPTIMIZATION OF THE ILLUMINA NEXTSEQ 500 SYSTEM FOR NEXT GENERATION SEQUENCING (NGS)-BASED SURVEILLANCE OF INFLUENZA Wednesday, 1st June 20:00 La Fonda Mezzanine (2nd Floor) Poster (PS‐2b.02) Background‐ Shoshona Le, Thomas Stark, Samuel Shepard, Elizabeth Neuhaus, David E Wentworth, John Barnes 1 Centers for Disease Control and Prevention The CDC Influenza Division (ID) currently utilizes Next Generation Sequencing (NGS) techniques to conduct genetic surveillance of circulating influenza virus strains. As a World Health Organization (WHO) Collaborating Center, ID receives between 8,000 and 10,000 specimen submissions per year for surveillance of influenza. ID has implemented a high throughput pipeline to meet this demand that is based on processing 96 influenza samples/controls per run across two Illumina MiSeq instruments, to generate all of the sequencing data used for influenza genetic characterization. For the Northern Hemisphere influenza 2015‐2016 season (October‐April), ID generated complete influenza genomes of over 3,500 viruses using this NGS pipeline, totaling more than 30,000 genome segments available for influenza analysis. During the peak of the influenza season, the high volume of influenza samples resulted in 12 MiSeq runs during a single month, with a large number of specimens still awaiting processing. The Illumina NextSeq 500 was procured in an effort to mitigate this issue, as it has 10‐15 times more data output of the current MiSeq in roughly the same run time. Because the NextSeq instrument differs significantly from the MiSeq instrument in the method of measuring fluorescence signals from the flowcell, assessments were conducted to determine whether the NextSeq would have any adverse effects on the influenza genetic data upon integration into the ID pipeline. Methods‐ To compare the data quality between the MiSeq and the NextSeq 500 instruments, libraries that had been prepared and previously run on the Miseq were re‐sequenced on the NextSeq, utilizing a loading workflow that was modified according to Illumina specifications. Pools of 96 samples that had been barcoded using an Illumina single index kit were run, then expanded to quad‐pools of 384 samples that had been barcoded utilizing Illumina Nextera A, B, C, and D 96‐index kits. Quality of the data and quantity of the reads were compared between the instruments, as well as consensus sequences, subpopulations, and coverage of the influenza genomes sequenced. Further comparisons between NextSeq v1 and NextSeq v2 chemistry were also examined. Conclusions‐ The NextSeq 500 System offers a cost efficient NGS‐based strategy for increasing the capacity of influenza NGS pipelines, and likely other viral pipelines, without sacrificing data quality or quantity. Its optimizable workflow, capability to provide a 10‐fold increase in data generation, and capacity to quadruple sample number per sequencing run, yet improve coverage enables high throughput sequencing to meet the demand for global surveillance of the influenza A and B viruses at less than half the cost of multiple MiSeq runs. 90
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Reliable solutions for focused NGS
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