Dr. Stephanie Rossow - Applications of Next Generation Sequencing

Appilcations of Next Generation Sequencing

Stephanie A Rossow DVM, PhDMinnesota Veterinary Diagnostic Laboratory

College of Veterinary MedicineUniversity of Minnesota

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.


• 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

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


• We want to know everything that’s in a sample.

• We’ll take that sample and send it to the lab.

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


• 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




• 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.


Unbiased Detection of Respiratory Viruses by Use of RNASequencing-Based Metagenomics: a Systematic Comparison to aCommercial 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.

Unbiased Detection of Respiratory Viruses by Use of RNASequencing-Based Metagenomics: a Systematic Comparison to aCommercial 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

Strain Variation and disease severity in congenital CMV infection – in search of a viral markerRavit Arav-Boger, MDJohns Hopkins University School of Medicine, 200 N. Wolfe St, Baltimore, MD 21287Ravit Arav-Boger: [email protected]

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.


• 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?


• “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?


• 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?


• 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.

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

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.

Dr. Stephanie Rossow - Applications of Next Generation Sequencing

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