High-Throughput Sequencing Technology Used for High-Resolution HLA Genotyping

New high-throughput sequencing technology has been developed to perform out high-resolution human leukocyte antigen (HLA) genotyping in research samples. The results of the study have important implications for future research on a wide variety of human diseases and tissue transplantation.

The study was published in October 2009 in the journal Tissue Antigens and reported that researchers at Roche Molecular Systems (Pleasanton, CA, USA) have used high-throughput sequencing technology from 454 Life Sciences (Branford, CT, USA), which is a Roche Company. In the study, the researchers performed ultra-deep amplicon sequencing of specific HLA class I and II gene loci with the Genome Sequencer FLX system and successfully assigned allele-level genotypes using software developed by Conexio Genomics (Perth, Australia).

HLA class I and class II genes play a vital role in the adaptive immune response. Importantly, they encode for the cell-surface proteins responsible for differentiating between self, non-self cells and other antigens. For example, accurate HLA genotyping is clinically important for hematopoetic stem cell (HSC) transplantation between unrelated donors and recipients to minimize the risk of graft rejection and graft versus host disease (GVHD). Accurate HLA genotyping is also critically important for research on many human diseases. "Specific alleles and haplotypes at the class I and class II loci are strongly associated with a variety of autoimmune disease as well as some cancers and infectious diseases,” explained Henry Erlich, study author and director of the department of human genetics at Roche Molecular Systems. "High-resolution, high-throughput HLA typing will be very valuable in these large research association studies.”

Accurate HLA genotyping is complicated by the highly polymorphic nature of this genomic region. There are hundreds of different allele sequences at the various HLA class I and class II genes. Current techniques, based on Sanger-sequencing technology, are limited in their ability to resolve "phase ambiguities,” which occurs typically, when an individual is heterozygous at many positions that are very close to one another.

"The challenge is to determine which polymorphic sequences go together on which alleles,” explained Mr. Erlich. "In our research, we were able to successfully assign allele-level HLA genotypes at eight loci in 48 individual samples from the data produced in a single Genome Sequencer FLX system run. The abundance of long sequencing reads allowed us to unambiguously assign HLA alleles in a much faster and more cost efficient manner than with traditional technologies. We were also able to detect rare variants, such as the nontransmitted maternal allele in a SCIDS [Severe Combined Immunodeficiency Syndrome] sample, reflecting the presence of maternal cells in the subject's circulation.”

In October 2009, 454 Life Sciences launched the latest GS FLX Titanium kits and software, offering increases in read length and throughput for amplicon sequencing. The new kits will improve targeting resequencing studies, such as HLA class I and II genotyping, by more completely covering loci with fewer amplicons and enhancing sensitivity for identifying rare variants and haplotypes. "Our early work with the GS FLX Titanium amplicon sequencing kits have demonstrated that we can now type more individuals per run and more exons per amplification reaction,” said Mr. Erlich.

"As proven by this study, 454 Sequencing Systems provides a cost-effective and reliable alternative to current research methods for HLA typing,” said Christopher McLeod, president and CEO of 454 Life Sciences. "This will only improve with the longer read lengths available in our latest series of GS FLX Titanium kits and software.”

Related Links:
Roche Molecular Systems
454 Life Sciences
Conexio Genomics