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Novel ambiguity resolution for HLA SBT methods
Abstracts
S31
3 37-P
NOVEL AMBIGUITY RESOLUTION FOR HLA SBT METHODS Steven Cate, Runying Tian, MaKenzie Roberts, William Hildebrand, Microbiology and Immunology, University of Oklahoma Health Science Center, Oklahoma City, OK, USA The Major Histocompatibility Complex (MHC) is recognized as the most polymorphic region of the human genome. Our Clinical HLA Typing Lab at the University of Oklahoma Health Sciences Center has performed over 17,000 Class I typings and 11,000 Class II typings in the past 8 years. In 2004, we procured an ABI 3700 that we have successfully sustained with a daily work load of 768 sequencing reactions for time constrained contracts. We have one by one identified and overcome various capacity constraints at points in the HLA Typing production process. The final capacity constraints are the numerous and diverse ambiguities that require resolution. Group Specific Sequencing (GSS) can be used to resolve ambiguous combinations resulting from certain allelic combinations of heterozygous sequence which arise in an average of 21% of Class I (n ⫽ 1500) and 15% of DRB1 samples (n ⫽ 500). GSS compared to the commonly used Group Specific Amplification (GSA) is an incredibly simple and reliable method with considerable cost savings. GSS will resolve a majority of ambiguities that arise from the combination of the 400 DRB1 different alleles due to a problematic ambiguity near the end of Exon 2. We have also used this technique to eliminate Group Specific Amplification steps in our DQA1 typing strategy. The final application is quick confirmation or elimination of Potentially New Alleles by analysis of homozygous sequence produced by GSS. The entire primary typing and secondary ambiguity resolution is accomplished with a single DNA sequencing platform and no additional PCR steps.
3 38-P
ALLELE-LEVEL CLASS I HLA TYPING BY SBT AND TARGETTED SSP Mary A. Portas, Michael T. Aubrey, Brian M. Freed, Histocompatibility, ClinImmune Labs, Aurora, CO, USA The pace of HLA class I allele discovery requires innovative strategies to maintain allele-level typings. We have developed a procedure, based upon commercial reagents, that stages sequencing followed by targetted SSP. This strategy minimizes DNA consumption, reagent costs, and turn-around-time while maximizing allele resolution. For this pilot study, DNA was extracted from N⫽17 bone marrow recipients with the GenoM6 robot and characterized by heterozygous sequence-based typing (SBT, Abbott Laboratories) on an ABI 3100 automated sequencer. If SBT alone achieved an allele-level typing we stopped further testing. For ambiguities outside the sequenced exons we applied sequence-specific AmbiSolv (Dynal Biotech) primer mixes. Routine class I serology sometimes ruled out null alleles. Samples with cis-trans ambiguities were resolved using subsets of Dynal Biotech high-resolution SSP Unitrays. Subsets were prepared by cutting the Unitrays into 8 to 12 individual 8-well strips. Although cutting the Unitrays increased handling and management, it mitigated DNA consumption and costs. The resolution at each locus was evaluated after SBT and also after application of secondary technologies. Allele-level typings (e.g., B*0801/B*4402) were defined as having two possible alleles per locus. Only 59%, 59%, and 47% of HLA-A,B,Cw typings, respectively, were allele-level by SBT; after targetted SSP, 100% of HLA-A,B,Cw typings were allele-level (see below table). Thus, a combination of SBT, serology, and targetted SSP routinely achieves allele-level typing. TABLE 1 Allele-Level Resolution After SBT and Targetted SSP HLA-A HLA-B SBT/Serology 59% 59% SBT plus AmbiSolv 65% 71% SBT plus 1 Strip 82% 82% SBT plus 1 Strip plus Ambisolv 88% 94% SBT plus ⬎1 Strip plus Ambisolv 100% 100%
HLA-Cw 47% 71% 65% 94% 100%
S31
3 37-P
NOVEL AMBIGUITY RESOLUTION FOR HLA SBT METHODS Steven Cate, Runying Tian, MaKenzie Roberts, William Hildebrand, Microbiology and Immunology, University of Oklahoma Health Science Center, Oklahoma City, OK, USA The Major Histocompatibility Complex (MHC) is recognized as the most polymorphic region of the human genome. Our Clinical HLA Typing Lab at the University of Oklahoma Health Sciences Center has performed over 17,000 Class I typings and 11,000 Class II typings in the past 8 years. In 2004, we procured an ABI 3700 that we have successfully sustained with a daily work load of 768 sequencing reactions for time constrained contracts. We have one by one identified and overcome various capacity constraints at points in the HLA Typing production process. The final capacity constraints are the numerous and diverse ambiguities that require resolution. Group Specific Sequencing (GSS) can be used to resolve ambiguous combinations resulting from certain allelic combinations of heterozygous sequence which arise in an average of 21% of Class I (n ⫽ 1500) and 15% of DRB1 samples (n ⫽ 500). GSS compared to the commonly used Group Specific Amplification (GSA) is an incredibly simple and reliable method with considerable cost savings. GSS will resolve a majority of ambiguities that arise from the combination of the 400 DRB1 different alleles due to a problematic ambiguity near the end of Exon 2. We have also used this technique to eliminate Group Specific Amplification steps in our DQA1 typing strategy. The final application is quick confirmation or elimination of Potentially New Alleles by analysis of homozygous sequence produced by GSS. The entire primary typing and secondary ambiguity resolution is accomplished with a single DNA sequencing platform and no additional PCR steps.
3 38-P
ALLELE-LEVEL CLASS I HLA TYPING BY SBT AND TARGETTED SSP Mary A. Portas, Michael T. Aubrey, Brian M. Freed, Histocompatibility, ClinImmune Labs, Aurora, CO, USA The pace of HLA class I allele discovery requires innovative strategies to maintain allele-level typings. We have developed a procedure, based upon commercial reagents, that stages sequencing followed by targetted SSP. This strategy minimizes DNA consumption, reagent costs, and turn-around-time while maximizing allele resolution. For this pilot study, DNA was extracted from N⫽17 bone marrow recipients with the GenoM6 robot and characterized by heterozygous sequence-based typing (SBT, Abbott Laboratories) on an ABI 3100 automated sequencer. If SBT alone achieved an allele-level typing we stopped further testing. For ambiguities outside the sequenced exons we applied sequence-specific AmbiSolv (Dynal Biotech) primer mixes. Routine class I serology sometimes ruled out null alleles. Samples with cis-trans ambiguities were resolved using subsets of Dynal Biotech high-resolution SSP Unitrays. Subsets were prepared by cutting the Unitrays into 8 to 12 individual 8-well strips. Although cutting the Unitrays increased handling and management, it mitigated DNA consumption and costs. The resolution at each locus was evaluated after SBT and also after application of secondary technologies. Allele-level typings (e.g., B*0801/B*4402) were defined as having two possible alleles per locus. Only 59%, 59%, and 47% of HLA-A,B,Cw typings, respectively, were allele-level by SBT; after targetted SSP, 100% of HLA-A,B,Cw typings were allele-level (see below table). Thus, a combination of SBT, serology, and targetted SSP routinely achieves allele-level typing. TABLE 1 Allele-Level Resolution After SBT and Targetted SSP HLA-A HLA-B SBT/Serology 59% 59% SBT plus AmbiSolv 65% 71% SBT plus 1 Strip 82% 82% SBT plus 1 Strip plus Ambisolv 88% 94% SBT plus ⬎1 Strip plus Ambisolv 100% 100%
HLA-Cw 47% 71% 65% 94% 100%