1. Christian Gabriel, Linz
Is next generation sequencing mature for medical diagnostics? Applications from immunogenetics to virology by 454 Sequencing 
Christian Gabriel, Linz

2. Red Cross Transfusion Service Linz
Die neue Blutzentrale Linz: ganz rechts das allgemeine Krankenhaus.
Blutzentrale Linz

3. Jumping out of biology
Medical applications

4. A very brief introduction
IMMUNOGENETICS

5. MHC 3 classes Important for: MHC I – bind TCR´s of CD8
The distinction of “non-self and self”
Antigen presentation
3 classes
MHC I – bind TCR´s of CD8
MHC II – bind TCR´s of CD4
MHC III - complement C2, C4, TNF
Many other functional proteins, which resemble the structure of MHC
Different levels of expression in different tissues.

6. Figure 5-16

7. new alleles Last lecture Spring 2008 in Switzerland IMGT/HLA Database
ImMunoGeneTics 7

8. Numbers of HLA Alleles HLA Class I alleles 4,383
HLA Class II alleles 1,291
HLA Alleles 5,674
Other non-HLA Alleles 8

9. HLA- A*02I101I01I02IL HLA nomenclature
Shows differences in the non-coding region
Shows synonymous subsitition in the coding region
Silent substitution
Locus
HLA- A*02I101I01I02IL
Allele group –
broad specificity
serologic equivalent
HLA-protein
Suffix : L, S, C, A, Q, N
N= null allele
L = Low cell surface expression
S = Secreted molecule but not present on the cell surface
C = In the cytoplasm but not on the cell surface.
A = indicates aberrant expression
Q = allele is questionable 9

10. MEDICAL USE

11. Medical significance of HLA
Transplantation
Bone marrow and stem cell transplantation
Solid organs: esp. kidney-transplantation
Association of HLA-antigens with diseases and predispositions:
Autoimmune diseases (Mb Bechterew)
Pharmacogenetics
Infectious diseases

12. Mismatch Stem cell transplantation
Donor
Allele Mismatch
Donor
Antigen Mismatch
Patient
MHC I HLA-A*0201,3101 HLA-B*4402,5601 HLA-C*0102,0501 MHC II DRB1*0101,0401 DQB1*0301,0501
MHC I HLA-A*0205,3101 HLA-B*4402,5601 HLA-C*0102,0501 MHC II DRB1*0101,0401 DQB1*0301,0501
MHC I HLA-A*2401,3101 HLA-B*4402,5601 HLA-C*0102,0501 MHC II DRB1*0101,0401 DQB1*0301,0501 12

13. The more mismatches you have, the worse is the outcome
Survival is more dependent on HLA-match the earlier you have your treatment.
Lee et al: Blood 2007 Dec 15;110(13):

14. Methods and Variations
Complex labs produce complex solutions
Methods and Variations

15. HLA Typing Methods: Resolution Serology: Molecular: Low = 2 digits
(complement-dependent-cytotoxity-assay)
Molecular:
PCR-SSO (sequence-specific oligonucleotides)
PCR-SSP (sequence-specific primers)
Sequencing (sequencing-based typing, SBT)
Resolution
Low = 2 digits
High = 4 digits
Ultra-high = all digits

16. Sequencing (sequencing-based typing, SBT)R M
Atria HLA B (generic sequencing)

17. Regions of interest
class I
class II

18. Time, work-load and ambiguities
PROBLEMS

19. Sequencing (sequencing-based typing, SBT)
vs.
B*1801,5502

20. Allele ambiguity example: HLA-B B*0702, 4402 B*0702, 4419N
outlier mutations: allele ambiguitiy
results when polymorphisms that distinguish
alleles fall outside of the regions
examined by the typing system
Exon 3
Exon 2
Exon 1
Exon 4
example: HLA-B
B*0702, 4402
B*0702, 4419N
Polymorphic positions
Core heterozygous sequence data

21. Genotype ambiguities A+B=D+E
Results from an inability to establish phase between closely linked polymorphisms identified by the typing system
example: HLA-B
B*0702, 4402
B*0720, 4416
B*0724, 4421
A+B=D+E

22. cis/trans Problems M = A und T S = G und C
TGGAGGGCSMGTGCGTGGA
S = G und C
M = A und T SM GA CT GT CA
TGGAGGGCSMGTGCGTGGA
Number of possible linkages = 2n
n=2; 4 combinations
n=4; 16 combinations

23. Workflow stem cell Transplantation
Diagnosis
Registration
1-6 m
No sibling
SBT Patient
Family typing
SSP, Serology
Donor requests,
typing
3-6 wks
selection
Selection and
donation
HLA- Typing
For confirmation
Collection
Conditioning
Transplantation
Transplantation

24. Ergebnisse: Es wurden 32 Proben mit ambiguities ausgewählt
Ergebnisse: Es wurden 32 Proben mit ambiguities ausgewählt. Alle Proben sind durch Familientypisierung und Sequenzierung bestätigt worden. Mit der MID-Technologie konnten sie gleichzeitig typisiert werden. Dabei sind auch zusätzliche Nukleotidaustausche gefunden worden, die als stille Mutationen beschrieben werden können.

25. Selected use for the 454 system in immunogenetics
Registry Typing
Low resolution 4 digit (intermediate resolution)
A,B,C,DRB1 und DQB Amplicons pts.
Price: US Euros, Linz
HSCT Typing
High resolution 4 digits Ambiguity-free
A,B,C,DRB1 und DQB Amplicons pts.
Price: 600Euros, Linz 240Euros

26. HLA Alpha Site Study
Goal Technical performance study to compare high resolution 454 sequencing with state of the art high resolution typing (Sanger) of relevant HLA loci
Investigators 8 experienced and new 454 users
Samples Samples submitted by the participants, 20 chosen, blinded
Primers 14 primer sets labeled with 11 MIDs for amplicon generation (1 set consists of 2 MTPs with total 319 oligonucleotides dried in micro titer plates)
Software GS Flx SW plus Conexio Genomics ATF

27. HLA Study Workflow Steps for high resolution / high throughput protocol – manual workflow
„MR“ - 2 plates OR „HR“ - 4 plates
Amplicon Generation
Samples: 20 DNA samples +2 neg. controls
PCR: 308 rx (14 primers x11 MIDs x 2 plates)
Purification
Quantification
Equimolar Pooling: 2 pools (sample 1-10; samples plus neg. controls)
GS FLX Chemistry
emPCR
Breaking
Enrichment
GS FLX Sequencing
PTP: 1 run; 2-regions; 10 samples (MIDs) / region
GS FLX SW: v2.0.01
Conexio ATF Software
Read length: 250 bp
Read depth: 670 reads/amplicon
acc. to standard 454 protocols

28. Genotype Assignment Assignment for 95% of the 2240 genotypes examined Causes for Failure to Assign Genotype 3%
4/8 sites had no genotypes omitted due to procedural/technical issues

29. Summary of Agreement and Concordance
Agreement = Identical ambiguity string obtained
Concordance = Reported genotype/allele in a limited ambiguity string matched submitted

30. Conclusions Improvements recommended by participants Workflow
Simplification (Titanium)
Automation
Additional ambiguity resolution esp. for null alleles (Ti UHR)
Conexio ATF software (will be addressed in next version)
Sequence insertions in new variants should be visible
Allow easier manipulation of less abundant sequences sometimes needed for genotyping
Allow program to automatically correct for specifically identified systematic sequencing errors in homopolymer run/end of sequence of a given amplicon

31. Acknowledgements

32. Haplotyping

33. Team Genomics
Johannes Pröll, Christa Hackl, Katja Hofer, Steffi Stabentheiner, Martin Danzer,
Norbert Niklas