Resolving Ambiguities

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Presentation transcript:

Resolving Ambiguities

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

Example HLA-C: Allele Ambiguity 3 3

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

Example HLA-C All ambiguities resolved ! 5 5

Genotype Ambiguity

Genotype Ambiguity

Genotype Ambiguity

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

Genotype Ambiguity 10 10

Genotype Ambiguity B16 B20 B26 3 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

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

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

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

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

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

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

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

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