Empowering genotyping by sequencing in animal applications - the power of partnering.

Transcription

1 Empowering genotyping by sequencing in animal applications - the power of partnering. Round 1: About cats, dogs and cattle Wim van Haeringen, PhD

2 Content Company Introduction Background including market analysis, goals Scope Results (examples) Future development Conclusions

3 Company background Established 1986 DNA is our core Wim van Haeringen PhD university Utrecht CEO of VHLGenetics since 2000

4 3 Business Units in Europe, Distribution network expanding

5 Fields of expertise Non-human DNA services Animal Plant Microorganisms

6 Portfolio Detection of genetic variants animals Parentage verification Diseases, mutations Detection of genetic variants plants Hybrid purity, Variety control Resistance Detection pathogens Species identification

7 Continuous need for improvement Transition in markets, change of B2B to B2C Increased focus on animal welfare and consumption

8 Transition in customer requirements Historically, one application Either genetic markers Or > 10,000 genetic markers Major requirement for flexible panels in the range of 100 5,000 genetic markers

9 Partnership for codevelopment (1) Partnering is the answer as technology development requires applications Specific information and in depth know how available & Continuous technology development Lead to long term codevelopment

10 Partnership for codevelopment (2) Application partner: Detailed market information and >30 years of applications. Requirements routine lab: Flexibility in marker numbers Flexibility in sample numbers Cost effective Technology partner: Thermo Fisher Scientific, AgriSeq GBS and AmpliSeq.

11 Explanation of technology NB: DNA extraction. Good quality DNA should result in good results. Requirements: concentration may be different between species and applications. Next slides: overview of the four-step AmpliSeq technology

12 Ion Torrent Semiconductor Sequencing Technology Each well in an Ion Chip performs a microsequencing reaction from a templated bead Nature 475, (21 July 2011)

13 Fast Direct Detection DNA Ions Sequence Nucleotides flow sequentially over Ion semiconductor chip One sensor per well per sequencing reaction Direct detection of natural DNA extension Millions of sequencing reactions per chip Fast cycle time, real time detection

14 Ion Workflow Overview AgriSeq Library Prep Ion Chef Template Prep Sequencing Data Analysis Day 1 Day 1-2 (overnight) Day 2 Day 2-3

15 Ion S5 (XL) Systems Scalability 5-80 million reads per run based on 3 different chips supported Combine up to 384 barcoded samples per chip Flexibility in applications Combination of multiple species / applications on same chip Different applications of same species on same chip Ion M Reads Ion M Reads Ion M Reads

16 Current projects Canine V1 Genetic Variants Feline V1 Genetic Variants Bovine V1 Parentage Verification

17 Materials, Methods Canis lupus familiaris (CanFam3.1) Chromosomes: 39 sequences (including X) Other: 3229 sequences (unplaced scaffolds and mitochondrion) Felis catus (Felis_catus_8.0) Chromosomes: 19 sequences (including X) Other: sequences (unplaced scaffolds and mitochondrion) Bos Taurus: well defined marker panel for PV 96-well AgriSeq workflow library prep was performed on each sample and all libraries were pooled onto a single Ion 540 chip for template prep and sequencing on the Ion Chef and Ion S5 XL.

18 Definitions Call rate Sample Call Rate is the percent of markers generating a genotype call for a specific sample. Used to see if there is any issue with samples. Marker Call Rate is the percent of samples generating a genotype call for a specific marker. Used to look at performance of each marker to see if there are any underperforming markers. If the mapped reads at a particular marker for a particular sample do not meet all of the acceptance criteria for the TVC, then the result for that position is listed as a No Call.

19 Example: Feline Panel V1 Results (1) Run metrics 62 Markers, 96 samples: Loading Statistics

20 Example: Feline Panel V1 Results (2) Run metrics 62 Markers, 96 samples: Fragment lengths

21 Example: Feline Panel V1 Results (3) Run metrics 62 Markers, 96 samples: Alignment and Raw accuracy

22 Call Rate % Feline Panel V1-99.8% Call Rate Average 99.8% overall call rate Sample ID Call rate for most feline markers was 100%; other markers had call rate > 97.9%

23 X Mean Coverage Feline Panel V1 - Mean Coverage Mean coverage is ~13,000 reads per marker for 96 samples Markers

24 Summary of current results Feline Panel V1 62 marker panel was tested with 96 samples v1 panel call rate is 99.8 % for 96 samples Canine Panel V1 161 marker panel was tested with 192 samples v1 panel call rate is >98% for 192 samples Bovine Panel V1 200 marker panel was tested with 96 samples v1 panel call rate is >98,5% for 192 samples -> robust performance

25 Short term developments Availability via our service labs (booth 127) Marketing & Sales through Thermo Fisher Scientific Panel expansion with relevant markers

26 Conclusions GbS AgriSeq(R)is a robust tool for our applications in the range of 100 5,000 markers. Results confirm: Flexibility in marker panels Flexibility in sample numbers Fast turn around times Low hands on time Routine lab implementation: Plug and Play

27 Any questions?

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