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HLA Guide

HLA Typing

Key Takeaways

PCR-SSP & Real-Time PCR

  • Method: Uses allele-specific primers to amplify target HLA alleles.
  • Real-Time Adaptation: qPCR monitors amplification in real time.
  • Pros:
    • Rapid turnaround (∼2–5 hours)
    • Simple setup
  • Cons:
    • Low to intermediate resolution (often antigen-level)
    • Potential ambiguities and limited allele coverage

PCR-SSO (Sequence-Specific Oligonucleotide Probes)

  • Method: PCR amplification followed by hybridization to a panel of allele-specific probes.
  • Pros:
    • Multiplex detection of many alleles
    • Higher resolution than PCR-SSP
  • Cons:
    • Complex interpretation due to overlapping probe patterns
    • May yield ambiguous results needing secondary tests

Sanger Sequencing (SBT)

  • Method: Direct sequencing (commonly of exons 2,3 for class I; exon 2 for class II).
  • Pros:
    • High-resolution, allele-level typing
  • Cons:
    • Labor-intensive and low throughput
    • Challenges with phasing heterozygous positions

Next-Generation Sequencing (NGS)

  • Short-Read NGS (e.g., Illumina)
    • Method: Long-range PCR/capture with fragmentation into short reads (150–300 bp) and in silico assembly.
    • Pros:
      • High per-base accuracy
      • High throughput with comprehensive coverage
    • Cons:
      • Short reads cannot span full HLA genes
      • Requires computational phasing; turnaround typically 1–3 days

Long-Read Sequencing

  • PacBio SMRT Sequencing
    • Method: Single-molecule real-time sequencing of full-length HLA gene amplicons (3–10 kb) with consensus (HiFi) reads.
    • Pros:
      • Full-length, phase-resolved alleles
      • Ultra-high resolution including structural variants
    • Cons:
      • Expensive instrumentation
      • Lower throughput and longer run times
  • Oxford Nanopore Sequencing (ONT)
    • Method: Real-time, long-read sequencing via nanopores; reads span entire HLA genes.
    • Pros:
      • Rapid (results in <6 hours)
      • Complete phasing and allele-level resolution
      • Portable and flexible
    • Cons:
      • Historically lower raw read accuracy (improving with new chemistries)
      • More complex data analysis and workflow

Deceased Donor HLA Typing: Nanopore vs. Real-Time PCR

  • Real-Time PCR:
    • Speed: ∼2 hours
    • Resolution: Low to moderate (antigen-level or two-field)
    • Workflow: Established, automated, robust for urgent use
  • Nanopore Sequencing:
    • Speed: ∼4–5 hours
    • Resolution: High-resolution, allele-level typing with complete phasing
    • Benefits: Enhanced donor–recipient matching (e.g., precise epitope matching)
    • Considerations: Requires optimized protocols and advanced data analysis; currently emerging in clinical practice

1. PCR-SSP (Sequence-Specific Primers)

PCR-SSP is a method that uses sets of sequence-specific primers to amplify HLA alleles of interest. Each primer pair is designed to bind only if a particular HLA allele (or group of alleles) is present in the DNA. A positive PCR amplification indicates the presence of that target allele, and a pattern of positive vs. negative reactions across many primer sets is interpreted to determine the HLA type. Traditionally, PCR-SSP results are detected by end-point analysis (e.g. agarose gel electrophoresis) to see which reactions yielded a DNA band. This method is fast (a few hours) and does not require sophisticated equipment, making it useful for low to intermediate resolution typing and situations requiring quick results (). However, it typically provides broad antigen-level or two-digit allele resolution and may yield ambiguous combinations that require further testing.
Real-Time PCR (qPCR) Adaptation: Modern PCR-SSP assays have been adapted to real-time PCR (qPCR) platforms to expedite and simplify detection. Instead of running gels, the amplification is monitored in real time through fluorescent dyes or probes, so positive reactions are detected immediately as they occur. This real-time PCR SSP approach (e.g. kits like LinkSeq) can return full HLA typing results in roughly 2 hours, about half the time of traditional gel-based SSP (which is ~4–5 hours) () (). The faster turnaround is especially valuable in urgent scenarios such as deceased donor organ allocation. Additionally, real-time detection reduces manual steps (no gel handling) and often includes built-in duplicate wells to avoid interpretation errors (). However, implementing qPCR-based SSP requires a dedicated real-time PCR instrument, which can be costly, and thus may be a barrier for some laboratories (). Despite this, the efficiency of qPCR-SSP has made it a good fit for many organ procurement organizations and labs handling time-sensitive HLA typing workloads ().

2. PCR-SSO (Sequence-Specific Oligonucleotide Probes)

PCR-SSO involves an initial PCR amplification of a gene (or region) using generic primers, followed by hybridization of the PCR product to a panel of sequence-specific oligonucleotide probes. These probes are immobilized on a solid support (such as membrane strips or bead arrays) and are designed to bind specific HLA allele sequences. After hybridization, a detection system (colorimetric on strips or fluorescent on beads) reveals which probes have bound to the PCR product. The pattern of reactive probes corresponds to particular HLA alleles. SSO methods can be high-throughput and are often used for intermediate-resolution typing. They rely on probe design and may not distinguish alleles that share the probed sequences (leading to some ambiguities). The process is more labor-intensive and time-consuming than SSP, typically requiring an overnight or multi-hour workflow, but it provides a broader allele coverage per assay. PCR-SSO was a common workhorse for HLA labs, especially with the introduction of reverse SSO on Luminex bead platforms, allowing multiplex probing in a single reaction. Resolution is usually at the allele group (two-digit) or intermediate (four-digit) level, depending on the probe panel (). Ambiguities or novel alleles often require reflex to another method (like SBT or NGS) for clarification.

3. Sanger Sequencing (SBT - Sequence-Based Typing)

Sanger sequencing was long considered the gold standard for high-resolution HLA typing. In SBT, specific HLA loci are PCR-amplified and then sequenced by the dideoxy (Sanger) method. Typically, exons encoding the antigen recognition site (especially exons 2 and 3 for class I, exon 2 for class II) are sequenced. The resulting nucleotide sequence is interpreted to determine the HLA allele. Sanger-based typing provides allele-level resolution (four-digit or higher) because it directly reads the DNA sequence of HLA genes. It can detect novel polymorphisms in the sequenced regions and distinguishes alleles that differ by even a single nucleotide. However, SBT is labor-intensive and low-throughput: each sample-locus combination must be separately sequenced, and analyzing phase (which nucleotide belongs to which allele in a heterozygote) can be challenging if alleles are heterozygous in the sequenced region. Phase ambiguity in Sanger is often addressed by sequencing both directions or additional exons, but this increases time and cost. Sanger sequencing has largely been supplanted by next-generation sequencing for routine high-resolution typing () (), but it is still used for resolving certain ambiguities or for confirmatory testing of unusual alleles. It remains a reliable method with well-characterized accuracy, though its throughput limitations make it impractical for large volumes or time-critical testing.

4. Next-Generation Sequencing (NGS) – Short-Read vs Long-Read Technologies

Next-generation sequencing has revolutionized HLA typing by enabling massively parallel sequencing of HLA genes. NGS-based HLA typing can achieve high resolution (allele-level typing beyond two-field) across multiple loci in a single run (). Two main categories of NGS are in use: short-read platforms (like Illumina and Ion Torrent) and long-read platforms (such as PacBio and Oxford Nanopore). Both approaches offer advantages for HLA typing, but they differ in read length, phasing capability, turnaround time, and equipment requirements.

4.1 Short-Read NGS (Illumina, Ion Torrent)

Short-read NGS platforms (e.g. Illumina MiSeq/NextSeq, ThermoFisher Ion Torrent) generate reads typically a few hundred base pairs long. For HLA typing, protocols often employ long-range PCR or targeted capture to enrich full-length HLA genes or exons, then sequence them in many small overlapping fragments. These short reads are high quality (low per-base error rates) and, with deep coverage and sophisticated assembly software, can reconstruct the HLA allele sequences. Short-read NGS provides a comprehensive view of HLA gene variation, covering both coding and non-coding regions (). It can detect novel polymorphisms and phase variants within the length of reads or read pairs. In fact, NGS (short-read) has largely replaced Sanger sequencing for high-resolution typing in many labs, due to its throughput and ability to sequence multiple loci simultaneously () ().
Using short-read data, allele determination algorithms assemble or align reads to reference HLA alleles. When designed properly, NGS can yield unambiguous allele assignments at greater than two-field resolution for most samples (). Notably, short-read NGS allows analysis of introns and untranslated regions, which can help identify null alleles or marker polymorphisms outside the antigen recognition site. Moreover, the depth of sequencing provides an internal check on allele balance (each allele in a heterozygote should have roughly 50% of the reads at that locus) (), aiding quality control.
However, short-read NGS has limitations related to its read length. HLA genes are long (up to >5 kb) and highly polymorphic, and short reads (150–300 bp) cannot span the full length of most HLA alleles. This means distant polymorphisms may reside on separate reads, making it challenging to determine if they are on the same haplotype (phase) (). As a result, some allele combinations remain ambiguous if the sequencing strategy doesn’t cover a distinguishing position in one contiguous read. For example, short reads confined to exons can miss differences in introns or phase relationships between variants in different exons. Indeed, although Illumina-based NGS dramatically reduces ambiguities compared to older methods, residual ambiguities can persist when polymorphisms are in cis/trans configurations that are not resolved by the read length (). In one analysis, an Illumina NGS method could not distinguish a critical amino acid difference in HLA-DPB1 because the two variant positions were on separate exons; the result was a pair of possible alleles differing by a single epitope (). These limitations underscore the need for careful assay design (e.g. covering key exons) or informatics phasing algorithms when using short-read data. They also motivate the use of long-read sequencing to fully phase HLA alleles.
Another practical limitation is turnaround time. Short-read NGS workflows, including library preparation, sequencing (which often runs for many hours), and data analysis, typically take 1–3 days for HLA typing (). In settings where batching samples is necessary for cost-efficiency, the wait time for results can be on the order of days, not hours (). This is acceptable for routine typing of transplant candidates or registry volunteers, but it is too slow for urgent cases like deceased donor typing. Thus, while short-read NGS offers high resolution and throughput, it is not ideal when immediate results are needed.

4.2 Long-Read Sequencing (PacBio and Oxford Nanopore)

Long-read sequencing technologies address many of the challenges posed by short reads. Platforms like PacBio Single-Molecule Real-Time (SMRT) sequencing and Oxford Nanopore Technologies (ONT) sequencing can produce reads thousands to tens of thousands of bases long, enough to span entire HLA genes (including exons, introns, and UTRs) in single contiguous sequences () (). By reading each DNA molecule in one piece, these methods allow direct phasing of distant variants and eliminate the need to assemble overlapping fragments. This capability greatly reduces ambiguities in HLA typing, since the full allele sequence is obtained and variants can be assigned to specific haplotypes () ().
PacBio SMRT Sequencing: PacBio uses single-molecule real-time detection of nucleotide incorporations to sequence DNA. For HLA, typically long-range PCR is performed to amplify the full gene (~3–10 kb), and then the amplicon is sequenced in its entirety by the PacBio instrument (). The PacBio system generates long reads with a higher raw error rate than Illumina, but it produces multiple reads of the same molecule which can be consensus-called to dramatically improve accuracy (HiFi reads) (). A key advantage of PacBio is the ability to sequence without fragmentation or short-read assembly, yielding each allele’s sequence as a continuous read. This enables detection of complex or structural variants (insertion/deletions, copy number variations) within the HLA region that might be missed or misassembled by short reads (). PacBio’s long reads also naturally provide haplotype phasing – for a heterozygous individual, reads can be segregated by allele, producing two phased sequences (one per chromosome). The result is ultra-high resolution: PacBio-based HLA typing can resolve alleles to full-length, 6-8 digit resolution, covering all exons and introns of up to 6 loci simultaneously (). This level of detail can identify novel allele variants and phased SNP patterns with precision () ().
Despite its technical strengths, PacBio sequencing has seen limited adoption in clinical HLA laboratories so far (). The drawbacks have been mostly practical: the instrumentation is large and expensive, per-sample cost can be high unless many samples are multiplexed, and the analysis pipelines (though improving) are not as turnkey as those for commercial short-read kits (). Additionally, until recently there were few commercial HLA typing kits for PacBio, meaning labs had to develop and validate their own methods, which is a barrier in a regulated clinical context (). As the technology advances and becomes more accessible (newer PacBio systems are higher-throughput and more cost-efficient), it’s expected that long-read sequencing could play a larger role in HLA typing, particularly for applications where its unique strengths (like comprehensive allele resolution or resolving complex ambiguities) are needed () ().
Oxford Nanopore Sequencing: ONT sequencing is another third-generation approach that detects DNA sequences by measuring the electrical current changes as a DNA strand passes through a nanopore. It similarly produces very long reads (potentially read lengths of tens of kilobases or even whole HLA gene regions) and can sequence DNA in real time. ONT platforms (MinION, GridION, PromethION) are notably compact and portable, with devices like the MinION being pocket-sized and usable in the field or at point-of-care (). For HLA typing, nanopore sequencing can achieve full allele resolution (four-field or higher) by reading through entire intron-exon structures, thereby directly phasing all polymorphisms in a gene (). Long nanopore reads drastically cut down the ambiguity because even distant SNPs within an HLA gene are observed on the same single molecule read () (). This eliminates the cis/trans phase uncertainty that can plague short-read results ().
ONT library preparation is relatively fast and does not require PCR amplification of targets (though PCR-based enrichment can be used); even when amplification is used, the library prep and sequencing can be done rapidly. A single MinION flow cell can sequence multiple HLA genes for multiple samples concurrently, and smaller flow cells like the Flongle allow cost-effective sequencing of one sample at a time. Turnaround times for ONT HLA typing have been reported at under 6 hours for a full class I and II typing of a sample (). In one demonstration, 24 indexed samples were successfully sequenced on one flow cell in less than 24 hours (), highlighting that the throughput can be scaled as needed.
One of the major advantages of nanopore sequencing is the real-time data streaming – as soon as the run starts, data begins to come off the device and can be analyzed, which means in theory you could stop the run once sufficient data is collected for a result. This is particularly useful in urgent scenarios (see next section on deceased donors). The portability and speed of nanopore devices, along with relatively low upfront cost, make them attractive for on-site HLA typing or use in smaller laboratories (). Nanopore technology also has the unique ability to directly sequence RNA or detect epigenetic modifications, though those are more experimental features in the context of HLA typing ().
Historically, ONT sequencing had a higher raw error rate compared to Illumina or PacBio, which initially raised concerns for clinical use in HLA typing (where accuracy is paramount). Early versions of the technology often had 10–15% error rates per base, especially around homopolymer regions. However, continuous improvements in nanopore chemistries (e.g. R10 pore updates) and base-calling algorithms (many employing deep learning) have significantly improved accuracy in recent years (). With high-accuracy basecalling and error-correction by increasing read depth, modern ONT HLA typing methods have demonstrated accuracy approaching that of other NGS platforms () (). For example, a recent high-resolution nanopore HLA typing protocol showed 100% concordance with standard typing methods for all alleles tested (). This progress is prompting a re-evaluation of ONT for clinical HLA applications (). It is increasingly plausible that nanopore sequencing can be used reliably for HLA genotyping, combining the benefits of long-read phasing with acceptable accuracy.
In summary, long-read sequencing technologies (PacBio and ONT) offer the ability to sequence HLA genes in full, providing phase-resolved, unambiguous allele identification. They contrast with short-read Illumina/Ion approaches by trading some raw read accuracy (which is mitigated by coverage or consensus methods) for the ability to span whole genes. Long-read methods are rapidly maturing and have already proven valuable in research settings and pilot clinical studies. As the cost and complexity continue to decrease, we can expect long-read sequencing to augment or even replace some short-read HLA typing workflows, particularly for resolving difficult allele ambiguities and improving typing in contexts like highly polymorphic multi-locus regions or complex variants () ().

5. Rapid HLA Typing for Deceased Donors: Nanopore Sequencing vs. Real-Time PCR

Deceased donor HLA typing presents a unique challenge: the HLA type of an organ donor must be determined very quickly (often within hours) to allocate organs to matching recipients. Traditional high-resolution methods like Sanger or Illumina NGS are too slow in this scenario (). Instead, laboratories have relied on rapid, lower-resolution methods such as PCR-SSP (sometimes in a real-time PCR format) or SSO to get results in a timely manner. These rapid methods typically provide antigen-level or two-field typings sufficient for basic donor-recipient matching, with the understanding that ambiguities or exact allele identities will be resolved later if needed.
Real-time PCR (rtPCR) for Donor Typing: As mentioned earlier, real-time PCR versions of SSP have been employed by many organ procurement organizations to speed up typing. A qPCR-based HLA typing kit can produce a donor’s HLA-A, B, C, DR, DQ, DP types in about 2 hours from sample to result (). For example, one study using a 384-well LinkSeq real-time PCR kit reported ~2 hour turnaround, compared to ~4–5 hours with older gel-based SSP kits (). In practice, this means a donor’s HLA profile can be known very shortly after the organ is procured, enabling transplantation coordinators to identify compatible recipients almost in real time. Real-time PCR assays typically target the most polymorphic exons of each HLA gene (those encoding the peptide-binding region) and use a panel of allele-specific primer/probe sets. The readout is a pattern of which wells amplified, interpreted by software to assign an HLA type. While this yields a low-to-intermediate resolution result (often one or two-field allele assignment, and sometimes remaining ambiguities), it is usually sufficient for making an allocation decision. The simplicity and speed of qPCR typing have made it a standard approach for deceased donors, and it has proven to fit well in the fast-paced workflow of OPO laboratories (). However, limitations include the inability to detect novel or unexpected alleles (since the assay only detects what it’s designed for) and occasionally ambiguities when two different alleles react with an overlapping set of primers. In some cases, supplemental testing or assumptions based on population frequency must be used to resolve those before finalizing the match. Still, the priority in deceased donor typing is speed, and real-time PCR delivers on that priority with technology accessible to most HLA labs.
Nanopore Sequencing for Rapid Typing: Recent advances in long-read sequencing, particularly using Oxford Nanopore, have opened the door to performing high-resolution, allele-level typing on deceased donor samples within hours () (). In 2020, De Santis et al. reported the first application of NGS-based HLA typing for deceased donor allocation using an Oxford Nanopore MinION device (). They developed a protocol (ONT-Rapid HR HLA) that amplified all 11 classical HLA loci in a single tube (using a long-range PCR assay kit) and sequenced the amplicons on a MinION with a rapid flow cell (Flongle) (). The entire process—from DNA to full high-resolution type for HLA-A, B, C, DRB1/3/4/5, DQB1, DPB1, DQA1—was completed in about 4 to 4.5 hours (). This is only marginally longer than the real-time PCR methods, but the crucial difference is the resolution of the results. The nanopore method provided allele-level (four-digit or higher) typing for all loci, fully phased, and was 100% concordant with the typings previously obtained on those donors by conventional SSO and Illumina/Ion Torrent NGS (). In fact, in some cases the nanopore sequencing achieved higher resolution than the routine methods, identifying allele variants that the other methods had reported as ambiguous (). This proof-of-concept demonstrated that it is feasible to get complete, high-resolution HLA genotypes of a deceased donor quickly enough to inform organ allocation.
The ability to have high-resolution donor HLA typing prior to transplant could be game-changing. It allows consideration of HLA eplet/epitope matching and more precise avoidance of donor-specific antibodies, which is especially beneficial for highly sensitized recipients () (). Under the current paradigm with real-time PCR, transplant centers often only know broad antigen mismatches at the time of organ offer, and finer allele mismatches (which can matter for antibody compatibility) are discovered later. Nanopore sequencing could provide that finer detail upfront. With donor allele sequences in hand, algorithms can predict immunogenic epitopes and help find the best recipient, potentially improving graft outcomes.
It’s important to note that using nanopore sequencing in this on-call setting requires certain resources: a portable sequencer (MinION/Flongle), reagents for rapid library prep, and computational tools for base-calling and HLA allele assignment in near-real-time. In the reported study, these elements were optimized to fit the deceased donor workflow. As of now, this approach is still emerging and not yet widely implemented; it has proven possible in a research or pilot context (), but broad adoption will depend on further streamlining the process and demonstrating reliability across many cases. The cost per donor for ONT sequencing (including consumables) and the need for skilled personnel to run and interpret the sequencing are considerations that labs will weigh against the current, well-established qPCR methods.
In summary, both real-time PCR and nanopore sequencing can meet the stringent time requirements of deceased donor HLA typing, but they offer different trade-offs in resolution and complexity:
  • Turnaround Time: Real-time PCR can produce results in ~2 hours, whereas nanopore sequencing protocols have achieved results in ~4–5 hours () (). Both are within the allowable window for donor allocation (usually organs can be preserved for a few hours), but qPCR retains an edge in speed.
  • Typing Resolution: Real-time PCR (PCR-SSP) provides low to moderate resolution (often allele group or two-field typing). In contrast, nanopore sequencing yields high-resolution, allele-level typing, resolving almost all ambiguities (). This means ONT can identify the exact allele (e.g. A01:01:01:01 vs A01:01:01:02), whereas qPCR might only tell that the donor is HLA-A01 (with some ambiguity on the specific allele).
  • Equipment and Workflow: qPCR-based typing requires a real-time PCR instrument and is largely an automated, closed system once the PCR plate is set up. It’s a familiar workflow in many HLA labs. Nanopore sequencing requires a MinION device (which is portable and relatively inexpensive) and involves DNA library preparation and post-sequencing computational analysis. The ONT workflow is more complex and currently demands more hands-on expertise in sequencing.
  • Data Analysis: The interpretation of qPCR SSP results is straightforward with vendor software and yields a definitive or limited list of allele possibilities. Nanopore data analysis involves base-calling the raw signal to DNA sequence, then aligning or querying the sequences against an HLA database to assign alleles. Automated pipelines exist, but analysis is inherently more involved than reading a PCR outcome.
  • Accuracy and Validation: Real-time PCR methods are well validated, and their accuracy is high for the allele groups they are designed to detect. Nanopore sequencing, with recent improvements, has shown excellent concordance with standard methods (), but labs will still need to validate it thoroughly, especially to ensure no critical allele dropouts or miscalls occur under time pressure. Early error-rate concerns are being mitigated by improved chemistries ().
  • Use of Results: A low-resolution typing from qPCR is sufficient for initial organ allocation and crossmatching; high-resolution sequencing results go further, enabling refined compatibility checks (e.g. epitope matching, virtual crossmatch at the allele level). The additional information from ONT could be leveraged if available, but current allocation systems are built around antigen-level mismatches. Policies and software would need updates to fully utilize high-res donor typing in real time () ().
Both approaches will likely continue to play roles in the near future. Real-time PCR offers a practical, rapid solution that is already in use for life-saving decisions. Nanopore sequencing represents a cutting-edge advancement that brings high-resolution typing into the timeframe of organ allocation. As the HLA field moves toward greater precision (e.g., considering allele-level mismatches and eplet compatibilities), the ability to get full allele information from deceased donors quickly will become increasingly valuable. Ongoing improvements in long-read sequencing and continued experience from pilot programs will determine how quickly methods like ONT typing are adopted alongside or in place of current qPCR-based strategies for deceased donor HLA typing. () ()