Use of Next Generation Sequencing for Whole Genome Analysis of Pathogens - Dr. Douglas Marthaler, Veterinary Diagnostic Laboratory, 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. Using Next Generation
Sequencing for Whole Genome
Analysis of Pathogens
Douglas Marthaler, PhD
Assistant Professor
University of Minnesota Veterinary Diagnostic Laboratory
Fangzhou Chen, Matthew Jarvis, Ham Lam, and Todd Knutson
Leman Conference
September 19th, 2016
St. Paul, Minnesota

2. NGS platforms
NGS, Amplified Single Molecule
Sequencing
• 454 Sequencing-Roche
– GS Junior System
– GS FLX+ System
• Illumina-Solexa
– HiSeq
– Miseq
• Applied Biosystems-Thermo
– SOLiD 5500 systems
• Ion Torrent –Thermo
– Personal Genome Machine
(PGM)
– Proton
Third Generation Sequencing,
Single Molecule Sequencing
• Helicos
• Pacific Biosciences
• Oxford Nanopore
Technologies
– GridlON System
– MinION

3. Amplified Single Molecule
Sequencing

4. Third Generation Sequencing,
Single Molecule Sequencing

5. Multiplexing

6. Genome Assembly: Concept
Complete genome sequences don’t come flying out
of the sequencing machines, instead,
Tiny genome “pieces” are produced.
Must “stitch” the pieces together.
This is known in genomics as “Assembly”.

7. Genome Assembly Tools: Hardware
Minnesota Supercomputing Institute
Multi-processors workstations
Connected to
MSI network
Supercomputers
12
3

8. Whole genome comparison of PEDV
strains

9. Song D, Park B. Virus Genes. 2012 Apr;44(2):167-75. doi: 10.1007/s11262-
012-0713-1. Epub 2012 Jan 22. Review.
PubMed PMID: 22270324.

11. Regions of diversity
• NSP2 and NSP3 function is ambiguous, but
might play a role in host antagonist activities

12. Recombination within US strains

13. PRRS investigations
Can whole genome sequencing
answer all the industries question?

14. Will WG sequencing solve all the
problems?
• Neurological case of PRRS
– Virus was introduced from another system
– ORF5 =100% nucleotide identity
– WG = 99.33% nucleotide identity (~100 nt and 19 aa changes)
• What’s causing neurological signs?
– Undiscovered protein, amino acid changes, ect?
– More basic research is needed to understand this relationship!

15. Will WG sequencing solve all the
problems?
• PRRS break in non-immunized herd
– Severe clinical signs in piglets (Ct values = 6-11)
– ORF5 =99.17% nucleotide identity to vaccine strain
– WG = 99.6% nucleotide identity (61 nt changes)
• Revertant from the vaccine strain
• Any PRRS strain can cause abortions and mortality during late
gestation!

16. Acknowledgements
Swine veterinarians for sending the samples and allowing me to
assist with trying to solve the problem. THANK YOU!
• My apologizes for the lengthy duration to receive results
Meet the research team and learn
more about other research projects
Marthalerlab.org