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

Round 1: About cats, dogs and cattle

Wim van Haeringen, PhD

Content

Company Introduction

Background including market analysis, goals

Scope

Results (examples)

Future development

Conclusions

Company background

Established 1986

DNA is our core®

Wim van Haeringen PhD university Utrecht

CEO of VHLGenetics since 2000

3 Business Units in Europe, Distribution network expanding

Fields of expertise

Non-human DNA services

Animal

Plant

Microorganisms

Portfolio

Detection of genetic variants animals Parentage verification

Diseases, mutations

Detection of genetic variants plants Hybrid purity, Variety control

Resistance

Detection pathogens

Species identification

Continuous need for improvement

Transition in markets, change of B2B to B2C

Increased focus on animal welfare and consumption

Transition in customer requirements

Historically, one application Either 1 – 100 genetic markers

Or > 10,000 genetic markers

Major requirement for flexible panels in the range of 100 – 5,000 genetic markers

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

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™.

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

Ion Torrent™ Semiconductor Sequencing Technology

Nature 475, 348–352 (21 July 2011)

Each well in an Ion Chip performs a micro-

sequencing reaction from a templated bead

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

Ion Workflow Overview

AgriSeq

Library

Prep

Sequencing Ion Chef Template

Prep Data

Analysis

Day 1 Day 1-2

(overnight) Day 2 Day 2-3

1 2 3 4

Ion S5™ (XL) Systems

Ion 520™

5 M Reads

Ion 530™

15-20 M Reads

Ion 540™

60-80 M Reads

• 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

Current projects

Canine V1 Genetic Variants

Feline V1 Genetic Variants

Bovine V1 Parentage Verification

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: 267606 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.

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”.

Example: Feline Panel V1

Results (1) Run metrics 62 Markers, 96 samples:

Loading Statistics

Example: Feline Panel V1

Results (2) Run metrics 62 Markers, 96 samples:

Fragment lengths

Example: Feline Panel V1

Results (3) Run metrics 62 Markers, 96 samples:

Alignment and Raw accuracy

Feline Panel V1 - 99.8% Call Rate

Sample ID

Call R

ate

%

Average 99.8% overall call rate

Call rate for most feline markers was 100%; other markers had call rate > 97.9%

Mean coverage is ~13,000 reads per marker for 96 samples

Feline Panel V1 - Mean Coverage

Markers

X M

ea

n C

ove

rage

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

Short term developments

Availability via our service labs (booth 127)

Marketing & Sales through Thermo Fisher Scientific

Panel expansion with relevant markers

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

Any questions?