Applications of Next Generation Sequencing - Dr. Stephanie Rossow, College of Veterinary Medicine, University of Minnesota, from the 2016 Allen D. Leman Swine Conference, September 17-20, 2016, St. Paul, Minnesota, USA. More presentations at http://www.swinecast.com/2016-leman-swine-conference-material

1. Appilcations of Next Generation
Sequencing
Stephanie A Rossow DVM, PhD
Minnesota Veterinary Diagnostic Laboratory
College of Veterinary Medicine
University of Minnesota

2. Applications of Next Generation Sequencing
• The questions are similar to the start of PCR and PRRSv sequencing.
• The applications seem very straightforward.
• Infectious disease is rarely straightforward
• Cause and effect was a lot easier in the past when we had insensitive
diagnostic tests.
• If that approach had worked we wouldn’t be having this conversation.

3. Applications of Next Generation Sequencing
•What we want
•Immediate actionable results
•Broad range of specimen types
•Broad diversity of the infectious
disease agents possibly present
within a single specimen
•The dynamic nature of the infectious
disease agents

4. Clinical Infectious Diseases® 2015;61(11):1695–702

5. Applications of Next Generation Sequencing
• We want to know everything that’s in a sample.
• We’ll take that sample and send it to the lab.

6. A case report demonstrating the utility of next generation sequencing in analyzing serial samples from the lung following an infection with influenza A (H7N9) virus. Yongfeng Hua,
Yan Zhanga, Xianwen Rena, Yingmei Liu, Yan Xiaoa, Li Li, Fan, Yang,Haoxiang Su, Feng Liu, Haiying Liu, Bin Cao, Qi Jin. Journal of Clinical Virology 76 (2016) 45–50

7. Applications of Next Generation Sequencing
• Who has what?
• Does it matter who you sample?
• There is a difference between a person and a barn full of pigs.

8. A case report demonstrating the utility of next generation sequencing in analyzing serial samples from the lung following an infection with influenza A (H7N9) virus. Yongfeng Hua,
Yan Zhanga, Xianwen Rena, Yingmei Liu, Yan Xiaoa, Li Li, Fan, Yang,Haoxiang Su, Feng Liu, Haiying Liu, Bin Cao, Qi Jin. Journal of Clinical Virology 76 (2016) 45–50

9. Applications of Next Generation Sequencing
• Who has what?
• Does it matter who you sample?
• There is a difference between a person and a barn full of pigs.
• When did they get it?
• It depends on when you sample
When you sample determines what you find.
• Is every pig in the barn at the same stage of disease?
• Yes, no, sometimes, I don’t know

10. A case report demonstrating the utility of next generation sequencing in analyzing serial samples from the lung following an infection with influenza A (H7N9) virus. Yongfeng Hua,
Yan Zhanga, Xianwen Rena, Yingmei Liu, Yan Xiaoa, Li Li, Fan, Yang,Haoxiang Su, Feng Liu, Haiying Liu, Bin Cao, Qi Jin. Journal of Clinical Virology 76 (2016) 45–50

11. Applications of Next Generation Sequencing
• Who has what?
• Does it matter who you sample?
• There is a difference between a person and a barn full of pigs.
• When did they get it?
• It depends on when you sample
When you sample determines what you find.
• Is every pig in the barn at the same stage of disease?
• Yes, no, sometimes, I don’t know
• What sample did you take?
• Serum, oral fluids, fecals, tissues
• What tissues
• What other systems do you need to measure?
• No organ is an island.

12. A case report demonstrating the utility of next generation sequencing in analyzing serial samples from the lung following an infection with influenza A (H7N9) virus. Yongfeng Hua,
Yan Zhanga, Xianwen Rena, Yingmei Liu, Yan Xiaoa, Li Li, Fan, Yang,Haoxiang Su, Feng Liu, Haiying Liu, Bin Cao, Qi Jin. Journal of Clinical Virology 76 (2016) 45–50

13. Unbiased Detection of Respiratory Viruses by Use of RNA
Sequencing-Based Metagenomics: a Systematic Comparison to a
Commercial PCR Panel
• In addition, untargeted metagenomics detected an additional 12 viruses that
were either not targeted by the RVP or missed due to highly divergent genome
sequences. Normalized viral read counts for untargeted metagenomics
correlated with viral burden determined by quantitative PCR and showed high
intrarun and interrun reproducibility. Partial or full-length viral genome
sequences were generated in 86% of RNA-seq-positive samples, allowing
assessment of antiviral resistance, strain-level typing, and phylogenetic
relatedness. Overall, untargeted metagenomics had high agreement with a
sensitive RVP, detected viruses not targeted by the RVP, and yielded
epidemiologically and clinically valuable sequence information.
• RVP = Respiratory Viral Panel
• Graf EH, Simmon KE, Tardif KD, Hymas W, Flygare S, Eilbeck K, Yandell M, Schlaberg R. 2016. Unbiased detection of
respiratory viruses by use of RNA sequencing-based metagenomics: a systematic comparison to a commercial PCR panel. J
Clin Microbiol 54:1000 –1007. doi:10.1128/JCM.03060-15.

14. Unbiased Detection of Respiratory Viruses by Use of RNA
Sequencing-Based Metagenomics: a Systematic Comparison to a
Commercial PCR Panel
• Metagenomics-based pathogen detection is especially powerful when
many diverse pathogens cause overlapping symptoms and when
molecular markers for drug resistance are known.
• Graf EH, Simmon KE, Tardif KD, Hymas W, Flygare S, Eilbeck K, Yandell M, Schlaberg R. 2016. Unbiased detection of
respiratory viruses by use of RNA sequencing-based metagenomics: a systematic comparison to a commercial PCR panel. J
Clin Microbiol 54:1000 –1007. doi:10.1128/JCM.03060-15

15. Strain Variation and disease severity in congenital CMV
infection – in search of a viral marker
Ravit Arav-Boger, MD
Johns Hopkins University School of Medicine, 200 N. Wolfe St, Baltimore, MD 21287
Ravit Arav-Boger: boger@jhmi.edu
Synopsis
The wide spectrum of congenital CMV disease and known differences in the biology and in vitro growth of CMV strains
continue to drive studies in search for specific viral genetic determinants that may predict severity of congenital CMV
disease. Several CMV genes have been studied in detail in congenitally-infected children, but the complexity of the viral
genome and differences in the definition of symptomatic disease vs. asymptomatic CMV infection continue to raise
questions related to what constitutes a pathogenic CMV strain. In addition, the prevalence and role of multiple CMV
strains as opposed to infection with a single strain in disease severity is debated. Although the new era of highly-sensitive
next generation sequencing assays may provide detailed information on multiple genetic loci, strict criteria for
differentiating between strains will be required. Identifying a viral marker (or combination of markers) for prediction of
disease outcome could have a major impact on pre-natal diagnosis and vaccine development. Large, well-controlled studies
with long-term follow-up and use of standardized high stringency molecular techniques will be required to move the field
forward. Genetic variation of host genes may play an adjunctive role in the outcome of congenital CMV disease.

16. Applications of Next Generation Sequencing
• Understanding Next Generation Sequencing results
• How do you perfrom a pathogenesis study when you can’t cultivate the
agent?
• How do you perfrom a pathogenesis study involving multiple agents?
• How do you perform a pathogensis study if you don’t know what to give who
and when to give it?
• If you can’t understand the sequence of events (pathogenesis), when do you
sample and what do you sample?
• If you can’t understand the sequence of events (pathogenesis), how do you
intervene?

17. Applications of Next Generation Sequencing
• “Rich clinical data linked to pathogen genotypes will permit
predictions of prognosis, virulence and drug susceptibility for active
infections” That is, we won’t be able to reproduce everything
experimentally and we’ll need detailed, recorded, stored,
retrievalable data in a consistent code.
• We need to start taking into consideration host variation.
• We need to know what normal is.
• Is normal the same everywhere?

18. Applications of Next Generation Sequencing
• How many diagnostic samples do we take?
• Can you make a diagnosis with one pig or one sample?
• Sample size to overcome individual variation?
• It depends what question you want to answer.
• How do pathogens transition?
• Can we use this knowledge for better secondary interventions”
• I’ll be sampling all of the time.
• Can we take before and after samples to see what is different?
• If everything is the same, then have the percentages of what’s present
changed?

19. Applications of Next Generation Sequencing
• None of these questions are new.
• We have an opportunity to answer some of these questions.
• Sometimes it might be easy.
• More likely it will be complicated and it will be frustrating.
• We still need to understand disease pathogenesis – who has what,
when did they get it, how do I find it and what does it mean?
• Lets learn from the past.
• Understand what question you’re asking.

20. References
• Goldberg B, Sichtig H, Geyer C, Ledeboer N, Weinstock GM. 2015. Making the leap from
research laboratory to clinic: challenges and opportunities for next-generation sequencing
in infectious disease diagnostics. mBio 6(6):e01888-15. doi:10.1128/ mBio.01888-15.
• How Next-Generation Sequencing and Multiscale Data Analysis Will Transform Infectious Disease
Management. Theodore R. Pak and Andrew Kasarskis, Clinical Infectious Diseases®
2015;61(11):1695–702
• Strain Variation and disease severity in congenital CMV infection – in search of a viral marker.
Ravit Arav-Boger, Infect Dis Clin North Am. 2015 September ; 29(3): 401–414.
oi:10.1016/j.idc.2015.05.009.
• Raszek MM, Guan LL and Plastow GS (2016) Use of Genomic Tools to Improve Cattle Health in
the Context of Infectious Diseases. Front. Genet. 7:30. doi: 10.3389/fgene.2016.00030

21. References
• A case report demonstrating the utility of next generation sequencing in analyzing serial samples from the
lung following an infection with influenza A (H7N9) virus. Yongfeng Hua, Yan Zhanga, Xianwen Rena, Yingmei
Liu, Yan Xiaoa, Li Li, Fan, Yang,Haoxiang Su, Feng Liu, Haiying Liu, Bin Cao, Qi Jin. Journal of Clinical Virology
76 (2016) 45–50
• Graf EH, Simmon KE, Tardif KD, Hymas W, Flygare S, Eilbeck K, Yandell M, Schlaberg R. 2016. Unbiased
detection of respiratory viruses by use of RNA sequencing-based metagenomics: a systematic comparison to
a commercial PCR panel. J Clin Microbiol 54:1000 –1007. doi:10.1128/JCM.03060-15.