1. Resolving Ambiguities

2. Three Kinds of Ambiguities
Alleles differ outside the region amplified and sequenced:
Allele ambiguities
Several heterozygous allele combinations can result in identical heterozygous sequences.
Genotype ambiguities
Alleles differ only in regions where the sequence of some other alleles are unknown
Lack sequence ambiguities 2 2

3. Example HLA-C: Allele Ambiguity3 3

4. HLA Class I Strategy 2 3 4 5 6 7 8
3’UTR 1
5’UTR
Amplification primer with QIAGEN Long Range Polymerase
Core Sequencing primer
Extended Region Sequencing primer 4

5. Example HLA-C
All ambiguities resolved ! 5 5

6. Genotype Ambiguity

7. Genotype Ambiguity

8. Genotype Ambiguity

9. × × Genotype Ambiguity            C*07:02:01:01 C*16:02:019

10. Genotype Ambiguity 10 10

11. Genotype Ambiguity B16 B20 B263 4 5 6 7 8
3’UTR 1
5’UTR
B16
B20
B26
Amplification primer with QIAGEN Long Range Polymerase
Core Sequencing primer
Group Specific Sequencing Primer 11

12. Genotype Ambiguity GSSP B20 + B26 + B16 All ambiguities resolved ! 12

13. Ambiguities & Unknown Sequences
Allele lacking sequences
Sample: S0XXX
Match:
DPB1*04:02,33:01
No genotype ambiguities
Allele ambiguities:
DPB1*04:02
: DPB1*105:01 Exon 3: 619
DPB1*04:02,33:01 or
DPB1*105:01,33:01 14

14. Ambiguities & Unknown Sequences
DPB1*04:02,33:01 or DPB1*105:01,33:01
DPB1*04:02 and 105:01 differ in exon 3
DPB1*33:01 exon 3 sequence unknown

15. Ambiguities & Unknown Sequences
Trace matches with two genotypes
Sample: S0XXX
Match:
DPB1*04:02,33:01
No genotype ambiguities
Allele ambiguities:
DPB1*04:02
: DPB1*105:01 Exon 3: 619
M = A & C

16. Genotype Ambiguity A*01:01:01:01,02:01:01:01 & A*02:36,36:04
Resolved by GSSP-362GG (A18)
One GSSP can resolved a number of ambiguous genotypes:
GSSP-362GG resolves many other ambiguities 17

17. Expected Genotype Ambiguities Based on allele frequencies and heterozygous sequencing
HLA-A: 81 %
HLA-B: 82 %
HLA-C: 81 %
HLA-DPB1: 60 %
HLA-DPA1: 24 %
HLA-DQB1: 34 %
HLA-DQA1: 0%
HLA-DRB1: 63 % (GenDx strategy: 17%) 18 18

18. HLA Class I Strategy 2 3 4 5 6 7 8
3’UTR 1
5’UTR
Amplification primer with QIAGEN Long Range Polymerase
Core Sequencing primer
Extended Region Sequencing primer: Resolving Allele ambiguities
Group Specific Sequencing Primer: Resolving Genotype ambiguities 19

19. How to Resolve a Genotype Ambiguity
Use the Dynamic Ambiguity Resolving Tool.
This feature, included within SBTengine® selectively proposes a Group Specific Sequencing Primer (GSSP) that is included in the SBTexcellerator extended box!
DART™ 20