1. Introduction to GENDX HLA typing products

2. The GENDX Mission
GenDx develops and markets innovative, CE marked diagnostic products, providing crucial information for patient-tailored treatments in and beyond transplantation therapy. 2

3. GENDX Company tests and services, analysis software and education
Develops and markets a comprehensive line of IVD
tests and services, analysis software and education
Located in Utrecht, Netherlands
Spin-off from University Medical Centre Utrecht
Founded in 2005
ISO 13485:2003 quality system
CE-IVD marked products 3

4. GENDX Products SBTexcellerator® Reagents, CE-IVD
Accelerating HLA typing by excellent performance
SBTengine® Software, CE-IVD
Powerful HLA-typing software using SBT data
NGS-go® Reagents, RUO
Amplification strategy for down stream NGS applications
NGSengine® Software, RUO
The Ultimate software tool for NGS HLA-typing 4

5. GENDX Products Custom laboratory service
HLA-typing service for research and diagnostics
Education
HLA-SBT Training School, ABHI accreditation
Production Facility
For small-medium scale production of DNA diagnostics
ISO certified 5

6. SBTexcellerator® Reagents, CE-IVD Accelerating High Resolution HLA Typing by Excellent Performance
Single PCR
Coverage of essential regions
# Class I; entire gene amplified
# Class II; exon 2 & 3 and beyond
Unsurpassed ambiguity resolving power
# 2nd Generation GSSP
# DARTTM in combination with SBTengine®
Automated handling of null alleles in combination with SBTengine®
Easy integration in your laboratory due to flexible kit design
Primers; Total Solution approach to type 50 DNA samples
CE-IVD 6

7. SBTengine® Software, CE-IVD Powerful HLA-typing software using SBT data
Highly automated, fast and accurate allele assignment
Easy-of-use through uncluttered user interface
GSSP Predicting System (GPS) for selecting GSSP’s upfront – pre typing info
Filtering options of crucial positions- saves analysis time
XML-based reports
Convenient network licensing available for multi-user sites
New allele reporting
Reagent independent
Automatic updates of software and libraries
Fast, knowledgeable customer support
CE-IVD 7

8. Sequencing Based Typing
High-resolution method
Uses all nucleotides to determine HLA alleles
No changes in strategy needed with IMGT/HLA update
Detection of new alleles
Limited number of reactions
CE-IVD 8

9. SBT Strategies Core typing: sequencing essential regions
HLA-A, B: Exons 2,3 and 4
HLA-C: Exons 2 and 3
DRB: Exon 2, DRB4 also exon 3
DQB1: Exons 2 and 3
DPB1: Exon 2 9 9

10. HLA Nomenclature HLA-A*02:01:01:01 HLA-A2 Serology
HLA-A*02 Allele group
main specificity/ serological equivalent
HLA-A*02:01 Allelic subtypes:
differences in 3rd and 4th digit = different protein
HLA-A*02:01:01 Non-coding variation
Differences in 5th and 6th digit = different nucleotides, identical protein
Silent variation
HLA-A*02:01:01:01 Variation outside coding regions
Differences in 7th and 8th digit = variation outside coding regions
Intron variation, splice sites, promotor 10 10

11. HLA Nomenclature Additional characters: N Null alleles HLA-A*24:09N
No expression
Premature stop codon
Splice sites
L Low expression: HLA-A*24:02:01:02L
S Secreted: HLA-B*44:02:01:02S
Q Questionable expression: HLA-A*32:11Q
Unconfirmed aberrant expression 11 11

12. SBT Strategy SBTexcellerator® Class I
Amplification primer with QIAGEN Long Range Polymerase
Core Sequencing primer
Extended Region Sequencing primer
Group Specific Sequencing Primer
CE-IVD 12

13. Example HLA-C SBTexcellerator®
Amplification primer with QIAGEN Long Range Polymerase
Core Sequencing primer
CE-IVD 13 13

14. HLA-C, Core SBTexcellerator® - SBTengine® Allele ambiguities
CE-IVD 14 14

15. HLA-C, Core + Extended SBTexcellerator® - SBTengine®
All ambiguities resolved !
CE-IVD 15 15

16. Example HLA-B, Core SBTexcellerator® - SBTengine® Genotype ambiguity
CE-IVD 16 16

17. Example HLA-B, Core + GSSP’s SBTexcellerator®
GSSP B20 + B26 + B16
All ambiguities resolved !
CE-IVD 17 17

18. HLA-A Strategy
CE-IVD

19. HLA-B Strategy
CE-IVD

20. HLA-C Strategy
CE-IVD

21. HLA-DRB1 Strategy
CE-IVD

22. HLA-DQB1 Strategy
CE-IVD

23. HLA-DRB3/4/5 Strategy
CE-IVD

24. Reagent Box Set-up SBTexcellerator®2 3 4 5 6 7 8
3’UTR 1
5’UTR
CORE
EXTENDED
FULL
CE-IVD 24

25. SBTexcellerator® Single PCR reaction per HLA locus
For HLA Class I coverage of the complete gene
For HLA Class II coverage of exon 2 (and exon3)
Primers provided for amplification and sequencing
Core regions:
Extended regions to resolve allele ambiguities
GSSP’s to resolve genotype ambiguities
CE-IVD 25

26. Next Generation Sequencing Typing
Features
One pass method
Limited ambiguities
No need for GSSP’s and or specific sequencing primers
Bulk method – pooling of samples
For research use only 26

27. NGS-go® Reagents Amplification strategy for down stream NGS applications
A reagent box contains amplification primers for:
HLA loci A, B, C, DRB1 and DQB1
Single PCR reaction per HLA gene
Sufficient reagent for amplication of 24 samples for all 5 loci
Amplification is performed with Qiagen LongRange PCR kit
Coverage of essential regions
# Class I ; entire gene amplified
# Class II; complete coverage of relevant coding regions,
extending beyond exon 2 & 3 where necessary
Complete exon readability
Amplicon can be used for amplicon sequencing with various available NGS platforms
For research use only 27

28. NGSengine® Software The Ultimate software tool for NGS HLA-typing
Saves Time
High percentage of analysed reads
Automated HLA locus identification
Typing of heterozygous samples in one pass
Crucial position toggling
Easy handling of missing data
Improves results
Clear quality statistics
Genotype references
Whole gene representation
Phasing visualisation
For research use only 28

29. NGSengine® Software The Ultimate software tool for NGS HLA-typing
Easy implementation
NGS platform independent! Optimized for data from Roche 454, Ion Torrent and MiSeq
One button analysis
For research use only 29

30. Custom laboratory service HLA-typing service for research and diagnostics
Research projects
New alleles
# Validation & submission
Registry typing
Confirmatory typing
Diagnostics
ISO certified
ISO certified 30

31. Education HLA-SBT Training School, ABHI accreditation
HLA-SBT Training Sessions - 1 day
Maastricht, The Netherlands,
Saturday 11th May Just prior to EFI
HLA-SBT Training School - 2½ day - Including wet laboratory practice
Utrecht, The Netherlands
March 2013
Register at:
ISO certified 31