4 Approaches to HLA Type using Next Generation Sequencing

Human leucocyte antigen (HLA) genes are among the most polymorphic in the entire genome. They are located on the short arm of chromosome 6 within the major histocompatibility complex (MHC) and play an important role in organ or hematopoietic stem cell transplantation. Donor – recipient matching prior to transplantation is performed by examining 6 genes (A, B, C, DP, DQ, DR) that encode HLAs to reduce risk of transplant rejection or graft-versus-host-disease (GVHD).

 While the current “gold-standard” for HLA typing is Sanger Sequencing, defining the phase of sequence motifs is difficult and genotype ambiguity increases with each database release. Next generation sequencing (NGS) offers many advantages including: the ability to provide nucleotide-specific reads across the entire genome, clonal amplification provides phase information, and the ability to thoroughly evaluate larger genomic regions (introns). Unresolved technical issues however have limited the implementation of NGS in accredited HLA laboratories. These include significant library preparation and processing times and the more complex bioinformatics analysis needed to generate an easily readable HLA typing report.

 The main approaches to prepare DNA template for HLA typing on NGS platforms include:

  1. Multiplex PCR to amplify target regions, including exons or introns. Advantage: After PCR, fragmentation and ligation are no longer required as sequencing primers are designed as past of the initial targeting primers.  Disadvantage: Primer design is difficult due to multiple polymorphisms, necessitating primer pools.
  2. Long range PCR of individual loci followed by fragmentation and ligation of sequencing adapters. Advantage: Longer regions can be targeted. Disadvantage: Fragmentation and subsequent ligation of sequencing adapters can be difficult.
  3. Sequence capture using oligo based hybridization to target regions >20 Mb. Advantage: This technique is similar to exome capture and well characterized. Disadvantage: Hybrid capture of shorter HLA regions or the MHC is less effective than larger segments.
  4. Sequencing of the entire genome (whole genome sequencing). Advantage: This is the least biased way to examine HLA regions of interest. Disadvantage: Data analysis and the requirement to extract sequences of interest from the entire genome is difficult and not ready for routine studies.

Generating an unambiguous HLA genotype is important and software customized to each of these 4 approaches now exists. Several commercial companies, including bioinformatics providers on Genohub have software to accept data in FASTA or FASTQ and output accurate HLA genotype results.

 Whether you’re just beginning your HLA typing work and are interested in library prep, sequencing and an analysis solution or if you’ve already extracted your data and now are looking for the right analysis to generate an unambiguous report, Genohub offers complementary HLA consultation and can match you with the right service provider. Service providers on Genohub have experience using the four template preparation methods described above and have the pipelines in place for your analysis. To get started, fill out our NGS project consultation form and we’ll contact you with our recommendations. 


2 thoughts on “4 Approaches to HLA Type using Next Generation Sequencing

  1. FYI: I’m getting a 404 on https://genohub.com/ngs-consultation/%20 the link to your consultation form. Not a customer, was just curious.

    Also “Primer design is difficult due to multiple polymorphisms, necessitating primer pools.” from #1, I don’t think that it’s actually difficult. There is certainly considerable polymorphism in most exonic regions of HLA, but the introns are pretty conserved. Yes there are some polymorphisms in there… but not so many that using a few different primers should make it “difficult”.


    • Hi Mary, we fixed that 404..how embarrassing. Thanks for pointing it out ! The difficulty lies in the length of the primers and the actual pooling. I agree though, #1 in our list is probably the easiest to perform. Do you regularly perform HLA typing ?


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