1. 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 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
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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 or Lasse Rasmussen
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 Nov 16;10(11):e doi: /journal.pone 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 Euro Surveill. 2015;20(39). doi: /1560-
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