- Email: [email protected]
2-OR: A Bioinformatic approach to ascertaining the rarity of HLA alleles
Tuesday, October 28, 2008 2:00 PM - 3:30 PM Genotyping of Histocompatibility Antigens: Keeping Pace ASHI Scholar 1-OR
HIGH-RESOLUTION, HIGH-THROUGHPUT HLA DNA TYPING BY 454 USING GSFLX SEQUENCING WITH ID TAGS. Gordon Bentley,1 Bryan Hoglund,1 Russell Higuchi,1 David Sayer,2 Damian Goodridge,2 Henry A. Erlich.1 1Human Genetics, Roche Molecular Systems, Alameda, CA, USA; 2 Conexio Genetics, Fremantle, Australia. Aim: Due to the patchwork pattern of HLA allelic sequence polymorphism, most HLA typing methods generate a set of possible genotypes due, in part, to difficulties in setting phase because, in most current methods for HLA typing, both DNA alleles are amplified and typed or sequenced simultaneously. Resolving these phase ambiguities can add significantly to the complexity of the HLA typing protocol. We have addressed this issue using the massively-parallel pyrosequencing technology developed by 454 Life Sciences (Margulies et al. Nature, 2008) to sequence 15 amplicons from 8 HLA loci. Methods: The 454 sequencing technology can clonally propagate and sequence in parallel millions of single DNA molecules. The long read-lengths obtainable with the 454/RAS GSFLX instrument (⬎250 nucleotides) make possible the unambiguous determination of phase among heterozygous DNA sequences of HLA alleles. Results: Here we demonstrate this ability to resolve phase ambiguities and sequence 15 exons from 24 individual DNAs in a single GSFLX run. The throughput is made possible by the use of sample specific ID tags that allow pooling of samples yet maintain the ability to assign sequences to a specific individual. All individuals had on the order of hundreds of bi-directional reads (sequences) per amplicon. We have incorporated an HLA typing software (Assign ATF, Conexio Genomics) that accurately assigns HLA genotypes by analyzing the output from the GSFLX for the 8 loci we interrogate: HLA-A,B,C, DRB1, DQA1, DQB1, DPA1, and DPB1. Conclusions: The GSFLX sequencing system with Conexio genotype assignment software is capable of providing unambiguous genotype typing for 8 HLA loci and 24 individuals in a single run.
2-OR
A BIOINFORMATIC APPROACH TO ASCERTAINING THE RARITY OF HLA ALLELES. Derek Middleton,1 Faviel Gonzalez,1 Steven G.E. Marsh.2 1N. Ireland H&I Laboratory, City Hospital, Belfast, Ireland; 2HLA Informatics Group, Anthony Nolan Research Institute, London, United Kingdom. Aim: As part of 15th International Histocompatibility Workshop we have organised a project to examine the rarity of HLA alleles. Methods: Data from several sources has been collected; (i) From the IMGT/HLA Database; the population from which the allele was originally sequenced, the number of cells sequenced for the allele and the number of labs who have independently sequenced this allele; (ii) Data from NMDP on the incidence of the allele being reported to that Registry; (iii) Data in 700 populations from the website www.allelefrequencies.net; (iv) Data received from individual labs who found the allele in their population, along with ethnicity, haplotype, method of detection. Results: Data for all known alleles has been successfully added. Individuals are able to interrogate the dataset according to their own criteria. For example it is possible to select alleles (either all alleles or alleles at a specific locus) which have been sequenced x times, are found in y populations on the website, have been found in z individuals in NMDP and have been found w times in individual labs. The choice of each criteria is available to the individual to perform his search. As an example from the total number of HLA-A alleles (n⫽ 650) 227 (34.9%) would only have been sequenced once,would appear on the website ⬍3 times, would be on NMDP registry ⬍2 times and not reported by any other laboratory. Conclusions: The data is freely and readily available on the website www.allelefrequencies.net is continually updated (newly discovered alleles are automatically added, as is new data on confirmation of sequences) The data should be useful to any laboratory in deciding which alleles need to be routinely determined in their population.
HIGH-RESOLUTION, HIGH-THROUGHPUT HLA DNA TYPING BY 454 USING GSFLX SEQUENCING WITH ID TAGS. Gordon Bentley,1 Bryan Hoglund,1 Russell Higuchi,1 David Sayer,2 Damian Goodridge,2 Henry A. Erlich.1 1Human Genetics, Roche Molecular Systems, Alameda, CA, USA; 2 Conexio Genetics, Fremantle, Australia. Aim: Due to the patchwork pattern of HLA allelic sequence polymorphism, most HLA typing methods generate a set of possible genotypes due, in part, to difficulties in setting phase because, in most current methods for HLA typing, both DNA alleles are amplified and typed or sequenced simultaneously. Resolving these phase ambiguities can add significantly to the complexity of the HLA typing protocol. We have addressed this issue using the massively-parallel pyrosequencing technology developed by 454 Life Sciences (Margulies et al. Nature, 2008) to sequence 15 amplicons from 8 HLA loci. Methods: The 454 sequencing technology can clonally propagate and sequence in parallel millions of single DNA molecules. The long read-lengths obtainable with the 454/RAS GSFLX instrument (⬎250 nucleotides) make possible the unambiguous determination of phase among heterozygous DNA sequences of HLA alleles. Results: Here we demonstrate this ability to resolve phase ambiguities and sequence 15 exons from 24 individual DNAs in a single GSFLX run. The throughput is made possible by the use of sample specific ID tags that allow pooling of samples yet maintain the ability to assign sequences to a specific individual. All individuals had on the order of hundreds of bi-directional reads (sequences) per amplicon. We have incorporated an HLA typing software (Assign ATF, Conexio Genomics) that accurately assigns HLA genotypes by analyzing the output from the GSFLX for the 8 loci we interrogate: HLA-A,B,C, DRB1, DQA1, DQB1, DPA1, and DPB1. Conclusions: The GSFLX sequencing system with Conexio genotype assignment software is capable of providing unambiguous genotype typing for 8 HLA loci and 24 individuals in a single run.
2-OR
A BIOINFORMATIC APPROACH TO ASCERTAINING THE RARITY OF HLA ALLELES. Derek Middleton,1 Faviel Gonzalez,1 Steven G.E. Marsh.2 1N. Ireland H&I Laboratory, City Hospital, Belfast, Ireland; 2HLA Informatics Group, Anthony Nolan Research Institute, London, United Kingdom. Aim: As part of 15th International Histocompatibility Workshop we have organised a project to examine the rarity of HLA alleles. Methods: Data from several sources has been collected; (i) From the IMGT/HLA Database; the population from which the allele was originally sequenced, the number of cells sequenced for the allele and the number of labs who have independently sequenced this allele; (ii) Data from NMDP on the incidence of the allele being reported to that Registry; (iii) Data in 700 populations from the website www.allelefrequencies.net; (iv) Data received from individual labs who found the allele in their population, along with ethnicity, haplotype, method of detection. Results: Data for all known alleles has been successfully added. Individuals are able to interrogate the dataset according to their own criteria. For example it is possible to select alleles (either all alleles or alleles at a specific locus) which have been sequenced x times, are found in y populations on the website, have been found in z individuals in NMDP and have been found w times in individual labs. The choice of each criteria is available to the individual to perform his search. As an example from the total number of HLA-A alleles (n⫽ 650) 227 (34.9%) would only have been sequenced once,would appear on the website ⬍3 times, would be on NMDP registry ⬍2 times and not reported by any other laboratory. Conclusions: The data is freely and readily available on the website www.allelefrequencies.net is continually updated (newly discovered alleles are automatically added, as is new data on confirmation of sequences) The data should be useful to any laboratory in deciding which alleles need to be routinely determined in their population.