Use of next generation Sequencing (NGS) at a National Reference Laboratory, Denmark 2015/16 Authors: Thea Kølsen Fischer, Ramona Trebbien, Jannik Fonager, Bente Andersen, Mille W Poulsen and Lasse Rasmussen Affiliation: National WHO Measles Reference Laboratory, Statens Serum Institut, Denmark Introduction Morbillivirus is a negative-sense ssRNA virus and a member of the Paramyxoviridae family. The genome is ~16 kb long, coding for 2 non-structural (V, C) and 6 structural genes (N, P, M, F, H, L). Currently 23 genotypes belonging to 8 clades have been identified. In 2015 in Denmark, 9 of 186 samples tested positive for Morbillivirus RNA with PCR and 687 samples were screened for the presence of IgM and/or IgG antibodies vs. Morbillivirus. All PCR positive samples were Sanger sequenced and genotyped according to WHO guidelines. As the Danish NRL at SSI currently is comparing the public health and scientific benefit versus costs and work load of using NGS in surveillance for all relevant viruses under surveillance, we applied this method on the morbillivirus samples from 2015. In this method-development study, we looked at the feasibility of using NGS to obtain full genome sequences from cultivated versus uncultivated viruses (ref 1, 2). Sequencing of cultivated and non-cultivated samples using NGS   Test results from Illumina (Mi-Seq) Next Generation Sequencing of the strongest (lowest CT values) non-cultivated (clinical) samples compared with NGS results from cultivated isolates. Standard morbilli sequence length: 450 bp (N-450) For example this picture should have 900 x 900 pixels H1-index Coverage: 97% Av. Coverage: 10 Gaps: 13 H1-04 Coverage: 100% Av. Coverage: 33 Gaps: none (cultivated) Av. Coverage: 400 NGS results (cultivated vs uncultivated) Av. Coverage: 244 Outbreak and sample overview Non-cultivated Travel-history to China Cultivated and uncultivated Cultivated Non-cultivated Cultivatede Figure 1. Origin and processing of the analyzed samples Our result demonstrate that the standard 450 bp long measles sequence might limit our ability to detect variability within the virus, and that outbreak investigation might have its limitations in terms of establishing country of transmission etc. Using the more sensitive NGS method, we have succeeded in developing a method which works particularly well for cultivated specimens, less well for clinical samples. Therefore a future plan for us as a NRL is to develop an NGS-based method which works well in clinical un-cultivated morbilli samples in order to decrease the workload (and time) and indirectly the costs associated with NGS.      Achievements, challenges and future plans RESULTS: Genotypic characterization and sanger sequencing of measles-cases 2015 Phylogeny of Measles-cases (n=9), Denmark 2015 Genotyping of the 9 measles-cases in 2015 resulted in detection of H1 (n=7) and D8 (n=2) The 450 bp sequences obtained through the standard Sanger sequencing approach did no allow for any sequence based discrimination between sequences from patients infected with measles in different countries (Figure 2). Acknowledgements Figure 2. See sample description on Figure 1 We thank all Danish regional Clinical Microbiology Laboratories for their support to the national surveillance of measles and supplying the NRL with all relevant information and samples. For more information contact: Thea Kølsen Fischer email: thf@ssi.dk or Lasse Rasmussen email: lara@ssi.dk References Penedos AR, Myers R, Hadef B, Aladin F, Brown KE. Assessment of the Utility of Whole Genome Sequencing of Measles Virus in the Characterisation of Outbreaks. PLoS One. 2015 Nov 16;10(11):e0143081. doi: 10.1371/journal.pone.0143081. eCollection 2015. Rasmussen LD, Fonager J, Knudsen LK, Andersen PH, Rønn J, Poulsen MW, Franck KT, Fischer TK. Phylogenetic and epidemiological analysis of measles outbreaks in Denmark, 2013 to 2014. Euro Surveill. 2015;20(39). doi: 10.2807/1560- font size 12