Dr. Kevin Lahmares - MinION Next-Generation Sequencing-based Routine Identification and Strain Typing of Porcine Reproductive and Respiratory Syndrome Virus
"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"
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Dr. Kevin Lahmares - MinION Next-Generation Sequencing-based Routine Identification and Strain Typing of Porcine Reproductive and Respiratory Syndrome Virus
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
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
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
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
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
29.
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
32.
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
Editor's Notes
$200 per shotgun metagenome; $50 per bacterial genome; <$5 per amplicon