Haplotype-specific extraction: a universal method to resolve ambiguous genotypes and detect new alleles - demonstrated on HLA-B.

Haplotype-specific extraction (HSE) allows the collection of individual alleles by separating diploid samples into their haploid components. The separation step is performed using magnetic beads in conjunction with allele-group-specific probes. The haplo-separated DNA samples can be directly typed with downstream applications such as sequence-specific oligonucleotide (SSO) typing, sequence-specific primer (SSP) typing, and sequence-based (SB) typing. Here we show that HSE permits the direct sequencing of an allele in its individual, separated state, including previously unknown alleles. Allele pair combinations that cannot be resolved by SSP, SSO, or generic SB typings can be unambiguously typed after the alleles are separated by HSE, which allows for new alleles to be easily detected without cloning. We show how HSE was performed to separate samples with locus-specific ambiguities in human leukocyte antigen (HLA)-B, which could not be resolved by means of generic SB typing for either sample. After haplotype-specific separation of the respective allele pairs, novel polymorphisms in the HLA-B*56 and HLA-B*44 alleles were clearly detected by SB typing.

New strategies for efficient typing of HLA class-II loci DQB1 and DRB1 by using Pyrosequencing.

The characterization of genetic risk factors for complex diseases located on chromosome-6 frequently requires human leucocyte antigen (HLA) genotyping of large patient cohorts. Currently available methods do not support high-throughput HLA typing beyond the major allele group level. We, thus, developed a high-throughput approach for the HLA-DQB1 and HLA-DRB1 loci that is based on Pyrosequencing. Pyrosequencing offers a higher degree of automation than direct sequencing or oligotyping. Using a dispensation order optimized for the particular HLA locus, rapid group typing and fine resolution can be achieved. We implemented the method for two important HLA loci--DQB1 and DRB1. The HLA-DQB1 typing method comprises the following steps: splitting the potential alleles after a generic polymerase chain reaction (PCR) amplification into groups with a first Pyrosequencing reaction and resolving the split allele groups by means of five further Pyrosequencing reactions. The HLA-DR gene family is known to be the most polymorphic one in the HLA class-II region because of a large number of DRB1 alleles. Because of this complex nature, HLA-DRB1 typing was performed by means of a combination of sequence-specific PCR typing and Pyrosequencing. HLA-DQB1 typing and HLA-DRB1 typing were performed successfully by using standard DNA samples with the help of known HLA genotypes and in a blind study by using the samples from the Deutscher Zell Austausch 2002 and 2003. The approach was optimized and was practically tested for genotyping in disease association studies. Our well-elaborated Pyrosequencing-based protocols offer a new alternative to the existing HLA class-II typing methods and represent a convenient and economic solution, a unique combination of high accuracy with high-sample throughput.