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147-P: High Resolution HLA Genotyping by Next Generation 454 GS-FLX Sequencing Employing Automated Amplicon Handling
Abstracts
S109
147-P
HIGH RESOLUTION HLA GENOTYPING BY NEXT GENERATION 454 GS-FLX SEQUENCING EMPLOYING AUTOMATED AMPLICON HANDLING. Cherie L. Holcomb,1 Suzanne Gelber,1 Bryan Höglund,1 Tom Chu,2 Henry A. Erlich.1 1Human Genetics, Roche Molecular Systems, Pleasanton, CA, USA; 2 Pharm Research and Early Development, Roche Palo Alto, Palo Alto, CA, USA. Aim: The 454 GS FLX massively parallel pyrosequencing system is capable of providing high resolution HLA genotyping for multiple individuals at multiple loci in a single run using the Conexio Genomics ATF software. Achieving high throughput can be challenging due to the number of handling steps, including amplicon cleanup, quality check, quantification, dilution and pooling, involved in amplicon preparation prior to sequencing. Methods: To achieve high throughput genotyping, we have used 14 PCR primer pairs (exon 2, 3, and 4 for class I, exon 2 for DPB1, DQA1, and the DRB loci, and exons 2 and 3 for DQB1) with 11 multiplex identifier (MID) tags to amplify HLA exons from individual samples. Use of MIDs allowed pooling of the amplicons generated from different individuals prior to the emulsion PCR step. Using a liquid handler for partially automated preparation of amplicon pools prior to sequencing, we have typed 64 DNA samples derived from whole blood. To achieve a balance in read depth for the various amplicons, we employed a variation in construction of pools in which we added 2-4 times the molar concentrations of amplicons generated by the primers for A (exon 4), B (exon 2), C (exon 3), and DRB (exon 2). Results: The clonal nature of this sequencing system reduces genotype ambiguities by allowing the assignment of “phase” to linked polymorphisms within the same amplicon. Typically, in a single run with a depth of 300 sequence reads per amplicon, we have run 22 samples in 5 days with resultant genotyping of at least 98.3% of the 176 loci interrogated. Conclusions: The application of automation as well as construction of amplicon pools which produce balanced numbers of sequence reads can likely be applied generally to increase throughput of 454 sequencing.
148-P
POPULATIONS. William Klitz,1 Loren Gragert,2 Maiers Martin,2 Enric Carreras.3 1School of Public Health, University of California, Berkeley, CA, USA; 2Bioinformatics, National Marrow Donor Program, Minneapolis, MN, USA; 3Spanish Marrow Donor registry, Barcelona, Spain. Aim: Spain was one of the first European cultures to explore and occupy land from across the world’s oceans, including several centuries of occupation in Mexico and the Philippines. One possible consequence of that period is admixture with local populations. Here we assess the hypothesis of Spanish admixture in Mexican and Filipino samples through the use of HLA-typed population stem cell donor registries, also using Japanese samples as an out group. Methods: Haplotype frequencies were estimated from the Spanish National bone marrow registry and for the other populations from the National Marrow Donor Registry in the United States at two- digit resolution of HLA A, B and DRB1. The similarity index If was used to gauge overall haplotype identity. Results: Spanish A-B-DRB1 haplotypes have a similarity fraction of 48% with Mexico, 13% with the Philippines and 11% with the Japanese. This fraction of European admixture in contemporary Mexicans has been seen in studies using other genetic systems. The low and slight difference in If values between the Filipinos and Japanese suggest two possibilities. On the one hand little or no Spanish admixture may be present in the Philippines. The second possibility is that the availability of higher resolution haplotypes from each population might reveal a signature of Spanish admixture in the Filipinos largely invisible here because of the low resolution typing. Conclusions: We conclude that even at less than full allelic resolution HLA variation, the haplotype data can provide an indication of historical admixture between Spain and its former colonies. Two avenues of additional research will be able to refine this initial effort: a) analysis of samples at four digit resolution and b) analysis of the Spanish sample by province, allowing a more precise consideration of population diversity and differential migration to the Spanish colonies.
S109
147-P
HIGH RESOLUTION HLA GENOTYPING BY NEXT GENERATION 454 GS-FLX SEQUENCING EMPLOYING AUTOMATED AMPLICON HANDLING. Cherie L. Holcomb,1 Suzanne Gelber,1 Bryan Höglund,1 Tom Chu,2 Henry A. Erlich.1 1Human Genetics, Roche Molecular Systems, Pleasanton, CA, USA; 2 Pharm Research and Early Development, Roche Palo Alto, Palo Alto, CA, USA. Aim: The 454 GS FLX massively parallel pyrosequencing system is capable of providing high resolution HLA genotyping for multiple individuals at multiple loci in a single run using the Conexio Genomics ATF software. Achieving high throughput can be challenging due to the number of handling steps, including amplicon cleanup, quality check, quantification, dilution and pooling, involved in amplicon preparation prior to sequencing. Methods: To achieve high throughput genotyping, we have used 14 PCR primer pairs (exon 2, 3, and 4 for class I, exon 2 for DPB1, DQA1, and the DRB loci, and exons 2 and 3 for DQB1) with 11 multiplex identifier (MID) tags to amplify HLA exons from individual samples. Use of MIDs allowed pooling of the amplicons generated from different individuals prior to the emulsion PCR step. Using a liquid handler for partially automated preparation of amplicon pools prior to sequencing, we have typed 64 DNA samples derived from whole blood. To achieve a balance in read depth for the various amplicons, we employed a variation in construction of pools in which we added 2-4 times the molar concentrations of amplicons generated by the primers for A (exon 4), B (exon 2), C (exon 3), and DRB (exon 2). Results: The clonal nature of this sequencing system reduces genotype ambiguities by allowing the assignment of “phase” to linked polymorphisms within the same amplicon. Typically, in a single run with a depth of 300 sequence reads per amplicon, we have run 22 samples in 5 days with resultant genotyping of at least 98.3% of the 176 loci interrogated. Conclusions: The application of automation as well as construction of amplicon pools which produce balanced numbers of sequence reads can likely be applied generally to increase throughput of 454 sequencing.
148-P
POPULATIONS. William Klitz,1 Loren Gragert,2 Maiers Martin,2 Enric Carreras.3 1School of Public Health, University of California, Berkeley, CA, USA; 2Bioinformatics, National Marrow Donor Program, Minneapolis, MN, USA; 3Spanish Marrow Donor registry, Barcelona, Spain. Aim: Spain was one of the first European cultures to explore and occupy land from across the world’s oceans, including several centuries of occupation in Mexico and the Philippines. One possible consequence of that period is admixture with local populations. Here we assess the hypothesis of Spanish admixture in Mexican and Filipino samples through the use of HLA-typed population stem cell donor registries, also using Japanese samples as an out group. Methods: Haplotype frequencies were estimated from the Spanish National bone marrow registry and for the other populations from the National Marrow Donor Registry in the United States at two- digit resolution of HLA A, B and DRB1. The similarity index If was used to gauge overall haplotype identity. Results: Spanish A-B-DRB1 haplotypes have a similarity fraction of 48% with Mexico, 13% with the Philippines and 11% with the Japanese. This fraction of European admixture in contemporary Mexicans has been seen in studies using other genetic systems. The low and slight difference in If values between the Filipinos and Japanese suggest two possibilities. On the one hand little or no Spanish admixture may be present in the Philippines. The second possibility is that the availability of higher resolution haplotypes from each population might reveal a signature of Spanish admixture in the Filipinos largely invisible here because of the low resolution typing. Conclusions: We conclude that even at less than full allelic resolution HLA variation, the haplotype data can provide an indication of historical admixture between Spain and its former colonies. Two avenues of additional research will be able to refine this initial effort: a) analysis of samples at four digit resolution and b) analysis of the Spanish sample by province, allowing a more precise consideration of population diversity and differential migration to the Spanish colonies.