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Is next generation sequencing
mature for medical
diagnostics?
Applications from immunogenetics to virology
by 454 Sequencing
Christian Gabriel, Linz
Red Cross Transfusion
Service Linz
Blutzentrale Linz
Jumping out of biology
MEDICAL APPLICATIONS
A very brief introduction
IMMUNOGENETICS
MHC
• Important for:
– 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.
Figure 5-16
new alleles
Last lecture
Spring 2008 in Switzerland
IMGT/HLA Database
ImMunoGeneTics
http://www.ebi.ac.uk/imgt/hla/index.html
Numbers of HLA Alleles
•
•
•
•
HLA Class I alleles
HLA Class II alleles
HLA Alleles
Other non-HLA Alleles
4,383
1,291
5,674
114
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
HLAprotein
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
MEDICAL
USE
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
Mismatch Stem cell transplantation
Patient
Donor
Allele Mismatch
Donor
Antigen Mismatch
MHC I
HLA-A*0201,3101
HLA-B*4402,5601
HLA-C*0102,0501
MHC I
HLA-A*0205,3101
HLA-B*4402,5601
HLA-C*0102,0501
MHC I
HLA-A*2401,3101
HLA-B*4402,5601
HLA-C*0102,0501
MHC II
DRB1*0101,0401
DQB1*0301,0501
MHC II
DRB1*0101,0401
DQB1*0301,0501
MHC II
DRB1*0101,0401
DQB1*0301,0501
• 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):4576-83.
Complex labs produce complex solutions
METHODS AND VARIATIONS
HLA Typing
• Methods:
– Serology:
• (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
Sequencing (sequencing-based typing, SBT)
R
Atria HLA B (generic sequencing)
M
Regions of interest
α1
α2
α3
5´
3´
270bp
SP
E1
E3
276bp
276bp
E2
E4
TM
CP
CP
E5
E6
E7
CP
E8
class I
3,5 kb
5´
3´
SP
E1
270bp
E2
282bp
TM
E3
E4
14,5 kb
CP
CP
E5 E6
class II
Time, work-load and ambiguities
PROBLEMS
Sequencing (sequencing-based typing, SBT)
163.2=Y
B*1801,5601
vs.
B*1801,5502
Allele ambiguity
outlier mutations: allele ambiguitiy
results when polymorphisms that distinguish
alleles fall outside of the regions
examined by the typing system
Exon 1
Exon 2
Polymorphic positions
Core heterozygous sequence data
Exon 3
Exon 4
example: HLA-B
B*0702, 4402
B*0702, 4419N
Genotype ambiguities
Results from an inability to establish phase between closely linked
polymorphisms identified by the typing system
A+B=D+E
example: HLA-B
B*0702, 4402
B*0720, 4416
B*0724, 4421
cis/trans Problems
TGGAGGGCSMGTGCGTGGA
S = G und C
M = A und T
TGGAGGGCSMGTGCGTGGA
Number of possible
linkages = 2n
n=2; 4 combinations
n=4; 16 combinations
-------------SM-------------------------GA--------------------------CT--------------------------GT--------------------------CA-------------IUB Code
Bases
K
S
G,T G,C
W
A,T
M
A,C
Y
R
C,T A,G
Workflow stem cell Transplantation
Diagnosis
Registration
1-6 m
Family typing
SSP, Serology
Donor requests,
typing
3-6 wks
selection
Selection and
donation
HLA- Typing
For confirmation
Transplantation
Collection
Conditioning
Transplantation
Selected use for the
454 system in immunogenetics
Registry Typing
Low resolution 4 digit (intermediate resolution)
A,B,C,DRB1 und DQB1 9-10 Amplicons 100-200pts.
Price: US 50-100Euros, Linz 30 - 70
HSCT Typing
High resolution 4 digits Ambiguity-free
A,B,C,DRB1 und DQB1 16-18 Amplicons 50-100pts.
Price: 600Euros, Linz 240Euros
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
HLA Study Workflow
Steps for high resolution / high throughput protocol – manual
workflow
„MR“ - 2 plates OR „HR“ - 4
plates
Amplicon Generation
GS FLX Chemistry

Samples: 20 DNA samples +2 neg. controls 

PCR: 308 rx (14 primers x11 MIDs x 2 plates)

Breaking

Purification

Enrichment

Quantification

Equimolar Pooling: 2 pools
(sample 1-10; samples 11-20 plus neg. controls)
emPCR
acc. to standard 454
protocols
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
Genotype Assignment
Assignment for 95% of the 2240 genotypes examined
Causes for Failure to Assign Genotype
Cause
# of
Genotypes
Omitted
# of Sites
Affected
New allele
24 (2%)
8
Plate seal failure in
gPCR
16
1
Amplicon not added to
7
pool
4
3%
forward sequencing
primer not annealed
4
1
Bias against B-2
reverse reads
6
1
4/8 sites had no genotypes omitted due to procedural/technical issues
Summary of Agreement and
Concordance
% Concordance with Known Variants
% Req'd
Locus
% Agreement
Genotype
Allele
Man. Ed.
HLA-A
HLA-B
89
93
91
96
94
98
13
15
HLA-C
DPB1
DQA1
94
99
100
94
100
100
97
100
100
4
3
5
DQB1
DRB1
DRB3,4,5
99
97
97
100
98
98
100
99
99
4
10
13
Overall
96
97
98
8
Agreement = Identical ambiguity string obtained
Concordance = Reported genotype/allele in a limited ambiguity string matched
submitted
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
Acknowledgements
454 Life Sciences –
Conexio Genomics
A Roche Company
B. Simen
L. Blake
M. Egholm
D. Goodridge
D. Sayer
CHORI
E. Trachtenberg
M. Ladner
S. White
CHOP
D. Monos
D. Ferriola
C. Lind
Red Cross
Transfusion Services,
Linz
C. Gabriel
J. Pröll
DKMS Life Sciences
R. Wassmuth
I. Böhme
Institute of Immunology
and Genetics,
Kaiserslautern
R. Klein
B. Thiele
Roche Applied
Science
G. Schmitz-Agheguian
Roche Molecular
Systems
H. Erlich
B. Höglund
Ch Holcombe
Stanford University
D. Tyan
M. Pando
M. Anderson
Haplotyping
Team Genomics
Johannes Pröll, Christa Hackl, Katja Hofer, Steffi Stabentheiner, Martin Danzer,
Norbert Niklas