The case for full-length sequencing for high-resolution HLA typing

Aug. 22, 2018

Advances in DNA sequencing have revolutionized many tests commonly used in or ordered by clinical laboratories, but the implications for HLA typing have been particularly impressive. This task, a complete analysis of a person’s human leukocyte antigen (HLA) genes, is critical for matching donors and patients prior to organ or hematopoietic stem cell transplantation.

The results of HLA typing are used to inform clinicians about histocompatibility between a patient and donor blood or organs. When there is a high degree of matching, patient outcomes are much better—from transplant success to overall survival times. A bad match, on the other hand, can put the recipient’s life in danger.

While HLA typing is essential, it is by no means simple. The HLA genes are some of the most polymorphic and complex in the entire human genome. Thousands of alleles are currently known, and novel variations are continually being discovered. They span a 3.6 Mb region of the genome on chromosome 6. Over time, scientists and clinicians involved in HLA typing efforts have added more markers to the process in an attempt to improve the accuracy of a match between donor and recipient. More recently, they have shown that fully sequencing the HLA genes instead of just scanning key regions to genotype them facilitates a more robust matching process.1

Technology, however, has been a limitation on the ability to resolve the HLA region.2 A new approach to HLA typing that uses long-read DNA sequencing has demonstrated improved resolution, higher accuracy, and more complete information. This method is rapidly enhancing the community’s understanding of the HLA region and providing data that should translate directly to more successful transplants.

Full-length HLA

It may seem obvious that fully sequencing the HLA genes rather than just looking for known variable regions would provide more information that could be used to match transplant donors and recipients.

The more we learn about HLA variation, the more important it becomes to represent it accurately. Today there are more than 16,000 known HLA alleles for the class I and class II genes in this complex.3 Just a few decades ago, there were only 100 known HLA antigens, a number that seemed remarkably high at the time. As technical resolution has improved, the number of potential classifications has soared. A single category once generated by serology tests, for instance, has been so fine-tuned that it is split out into 700 different subtypes today.

The increasing resolution from sequence-based typing has led to more successful transplant outcomes. In a report from scientists at Anthony Nolan, a UK charity that launched the world’s first bone marrow registry in 1974, transplant patients matched with ultra-high resolution had 20 percent better probability of survival after five years compared to patients matched with lower resolution.4

Ideally, high-resolution views of the HLA genes would also factor in phasing information to help experts understand whether detected variants occur on the same or different chromosomes. Phasing variants would add a new level of precision to HLA typing, but it cannot be done with the short reads produced by most NGS or Sanger platforms when
variants are more than a few hundred bases apart.

Long-read sequencing

Long-read sequencing technology, the latest entry to the DNA sequencing realm, has a number of features that make it a compelling fit for HLA typing. These systems generate individual reads that are tens of kilobases long, making it possible to capture full-length class I and class II genes in single, continuous reads for a complete sequence. Because the systems work with single molecules, the long reads can phase even distant variants.

Labs that specialize in HLA typing have begun to adopt long-read sequencing and results indicate that this method produces higher-resolution, highly accurate data that could dramatically improve the process of matching transplant donors and recipients. At one lab for instance, scientists typed more than 500,000 samples for the National Marrow Donor Program’s “Be the Match” registry. Thousands of novel variants were detected, including hundreds that could cause null expression or alternative expression of HLA genes.5

Looking ahead

The clear advantages of long-read sequencing to generate full-length HLA gene sequences suggest to many in the industry that this approach should be the new standard for HLA typing. Going forward, the use of exon-based genotyping methods or short-read sequencing methods to analyze only pieces of genes or known variants might be considered inadequate for high-resolution HLA typing and be reserved for specific cases in which DNA quantity and quality are not amenable for long-read sequencing. In the interest of giving patients the best outcomes, clinical labs that outsource their HLA typing to reference labs could ensure that results are being generated with long-read sequencing. Now that it is possible to completely sequence and phase HLA genes for the most comprehensive view of this important genomic region, lab leaders should consider offering this technology to enhance patient care in a critical medical situation.


1. Mayor N, Robinson J, Alasdair JM, et al. HLA typing for the next generation. PLoS One. 2015. DOI:10.1371/journal.pone.0127153.
2. Hosomichi K, Shiina T, Tajima A, Inoue I. The impact of next-generation sequencing technologies on HLA research. J Hum Genet. 2015;60:665–673; .
3. HLA Alleles Numbers (online database). Nomenclature. 
4. Mayor N. Better HLA matching as revealed only by ultra-high resolution HLA typing results in superior overall survival. 32nd European Immunogenetics and Histocompatibility Conference, Venice, Italy.
5. Cereb N. New Horizons in HLA typing and haplotyping: revolution in HLA genomics. 32nd European Immunogenetics and Histocompatibility Conference, Venice, Italy.