"MinION Next-Generation Sequencing-based Routine Identification and Strain Typing of Porcine Reproductive and Respiratory Syndrome Virus - Dr. Kevin Lahmares, Virginia Tech, from the 2017 North American PRRS/National Swine Improvement Federation Joint Meeting, December 1‐3, 2017, Chicago, Illinois, USA. More presentations at http://www.swinecast.com/2017-north-american-prrs-nsif-joint-meeting"

1. Kevin Lahmers, Michelle Todd, James Stanton, Tanya LeRoith
MinION Next-Generation Sequencing-
based Routine Identification and
Strain Typing of Porcine Reproductive
and Respiratory Syndrome Virus

2. “Next Generation Sequencing”
• Parallel sequencing of multiple
fragments
• Short reads
• Long reads- single molecule reads

3. Applications of parallel seq
• Whole genome/exome sequencing
• RNAseq
• Targeted sequencing
 PCR
 Enrichment
• Metagenomics
 16S
 Shotgun

4. Short read technology comparisons
Technology Illumina 454 IonTorrent SOLiD
Detection Sequence
termination
Light
(pyrophosphate
release)
pH change Fluorescence
Advantages High accuracy
Low sequencing
cost
Fast run time Short run
time
Low cost
Highest
accuracy
Disadvantages Long read time
Short reads
Expensive
Low throughput
High error
rate
Expensive
Longer run
times

5. Long read technology comparisons
Technology Pacific Biosciences Oxford Nanopore
Detection Optical/fluorescence Electrical current
Advantages High accuracy
Kinetic analysis
Fast run time
Long reads
Inexpensive
Selective sequencing
Disadvantages Long read time
Equipment cost
Accuracy
Changing technology

6. Oxford Nanopore Technologies- MinION

10. Cost
• ‘Free’ sequencer
• ~$100 for library prep
• $500 per flow cell
 Reusable flow cell
 Barcoding for multiplexing
 Sequential sequencing

11. Minion Characteristics
• Long reads
 Average 8KBase
 Long 1 MBase
• 450 bps
• Up to 30 GBases per flowcell
• As little as 10 minutes for library prep
• Sequence data available immediately

12. Minion Characteristics
• Direct RNA sequencing
• Read until- Negative and positive selection of
sequence while reading
• Cas9

13. Hypothesis
• Direct shotgun metagenomic sequencing will
identify and genotype Porcine Reproductive
and Respiratory Syndrome Virus
 Genotyping as part of the process
 Identification of other pathogens concurrently

14. Read length

15. Read length

16. Read identity

17. Assembly identity

18. Workflow
RNA
isolation
cDNA
preparation
Adapter
ligation
Sequencing
Analysis
1 hour 2 hours 2 hours
Runs up to 48 hours
Sequence produced immediately
10 minutes
10 days
Or a lifetime

19. Bioinformatic approach
• MinKNOW software for sequencing
• Albacore 2.1.3 for basecalling
• Centrifuge- for shotgun metagenomic
analysis
 Custom databases- eg 781 PRRS complete
genomes
• Pavian for visualization of metagenome
• Blast sequences for confirmation

23. Initial testing
• Porcine Circovirus
 PCV 1/2a virus stock
• PRRSV
 VR2385 stock
 MN184B stock

24. Serum- 5 days post infection
• Experiment 1
 Roughly 400,000 reads per sample
 First PRRS positive with in 5 minutes
 32 reads to PRRS
 24 unique reads to VR2385
• Experiment 2
 19 reads to PRRS
 5 unique reads to MN184B

25. Serum
• Experiment 3
 178 reads to PRRS
 67 unique reads
• SD98-163_P83 18
• 1692-98 8
• P129 7
• SDU73 4

27. Sequence identity with NADC20
orf5• P129 97.7%
• SD98-163_P83 96.4%
• SDU73 96.2%
• 1692-98 95.0%
• VR2385 93.4%
• MN184B 89.7%

28. Three strains combined for
metagenomic analysis
• Combined read data from three separate runs
 233 hits
 Unique hits to:
• VR2385 - 24
• MN184B - 7
• SD98-163_P83 - 18

30. Conclusions
• Identification and genotyping is possible
• Have started barcoding samples to run multiple samples in
the same library
 Throughput
 Cost effective
• Analysis pipeline
 Must basecall separately because of amount of sequence
 ‘Centrifuge’ database created with over 700 full PRRS genomes
on NCBI
• Have identified other organisms concurrently

31. Other current applications at ViTALS
• Bacterial genome closure- WGS
• Amplicon sequencing for longer reads
• Vector sequencing
• Methylation analysis
• Direct RNA sequencing

33. Acknowledgements
• Virginia Tech
 Michelle Todd
 Dr. XJ Meng
 Dr. Tanya LeRoith
 Robert Settlage
• University of Georgia
 Dr. James Stanton
• University of Nottingham
 Dr. Matthew Loose
• Smithfield Farms
 Dr. Joe Fent
 Dr. Jeremy Pittman
• National Pork Board