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Views: 0 Author: Site Editor Publish Time: 2026-08-03 Origin: Site
HLA typing methods differ in the way they detect alleles, the amount of sequence information they generate, the number of loci they can process, and the level of laboratory infrastructure they require. A method that works well for a rapid, targeted HLA test may not be suitable for a high-resolution transplantation workflow or a multi-locus research panel.
The main options include PCR-SSP, PCR-SBT, Sanger sequencing, next-generation sequencing (NGS), and third-generation sequencing (TGS). These methods should not be selected only by instrument availability or the highest possible resolution. The better approach is to match the method with the assay’s intended use, target loci, required resolution, throughput, turnaround time, sample type, interpretation requirements, and available technical resources.
For laboratories developing or evaluating an HLA assay, HLA reference standards can provide characterized materials for comparing the expected genotype with the result generated by a selected workflow.
PCR-SSP is suitable for rapid, targeted detection when the relevant allele groups are already known.
PCR-SBT is a sequence-based workflow that commonly uses Sanger sequencing after PCR amplification.
Sanger sequencing is useful for focused locus or amplicon analysis but may produce ambiguity when HLA diversity exceeds the sequenced region.
NGS is generally better suited to multi-locus testing, higher sample throughput, and broader sequence coverage.
TGS can provide long-read information that helps resolve phasing and full-gene ambiguity.
No method is universally best. The appropriate choice depends on the assay’s intended output and operating conditions.
PCR-SBT and Sanger sequencing are closely related, but they are not exactly the same term.
PCR-SBT means polymerase chain reaction sequence-based typing. In this workflow, the target HLA region is first amplified by PCR and then sequenced to identify sequence variations. Sanger sequencing is one of the most common sequencing technologies used as the readout method for PCR-SBT.
Therefore, a laboratory may describe an assay as “PCR-SBT” when referring to the complete HLA typing workflow, or as “Sanger sequencing” when referring specifically to the sequencing technology. This distinction is important when comparing methods.
PCR-SBT describes the assay design and workflow. Sanger describes the sequencing platform or chemistry used to read the amplified DNA.
CB-Gene’s single-locus HLA genotyping standards are relevant to laboratories evaluating focused HLA targets using PCR-SBT or Sanger-based workflows.
PCR with sequence-specific primers, or PCR-SSP, uses primers designed to amplify only when a particular sequence or allele group is present. The result is usually determined by whether a specific amplification product is detected.
PCR-SSP does not normally read the complete HLA sequence. Instead, it answers a defined question: whether the target sequence recognized by a primer set is present in the sample.
PCR-SSP may be suitable for:
Rapid screening of known HLA alleles or allele groups
Low- to moderate-throughput testing
Laboratories with limited sequencing infrastructure
Assays requiring a defined yes-or-no result
Research workflows focused on selected HLA targets
PCR-SSP can be relatively straightforward to establish because it does not require a sequencing instrument or complex sequence-analysis pipeline. It can also provide fast results when the assay uses a limited number of primer reactions.
For a focused test, PCR-SSP may be more practical than sequencing because the laboratory only needs to determine whether the designed target is detected.
The main limitation is that PCR-SSP can only detect what the primer design is intended to recognize. Rare or unexpected variants may affect primer binding and lead to allele dropout, false-negative results, or results that require additional confirmation.
As the number of target alleles and loci increases, the number of primer reactions may also increase. This can make the workflow more labor-intensive and less convenient for comprehensive or high-resolution HLA typing.
PCR-SBT amplifies a selected HLA region and then determines its nucleotide sequence, commonly using Sanger sequencing. Compared with PCR-SSP, it provides direct sequence information rather than only a presence-or-absence amplification result.
The sequence is compared with reference sequences in an HLA allele database to assign the most appropriate allele or allele group. The final resolution depends on which regions are amplified and sequenced, the quality of the sequence data, and whether the observed sequence uniquely identifies one allele.
PCR-SBT is often a good fit for:
Single-locus or limited-locus HLA typing
Targeted assay development
Confirmation of selected alleles
Small laboratories with established PCR and Sanger facilities
Method comparison using defined reference materials
Workflows where sample volume is limited
PCR-SBT provides more sequence information than PCR-SSP and can identify sequence differences within the amplified region. It is useful when a laboratory needs more detail than a targeted primer reaction can provide but does not require a large multi-locus sequencing workflow.
It may also be easier to interpret than a high-throughput sequencing workflow when only a small number of loci or amplicons are tested.
PCR-SBT only evaluates the regions included in the assay. If two HLA alleles share the same sequence across the tested region, the result may remain ambiguous. Additional sequencing, supplementary primers, or another typing method may be required.
Heterozygous sequence traces can also contain overlapping peaks. In addition, PCR amplification design, allele dropout, nonspecific amplification, and phase ambiguity may affect interpretation.
Sanger sequencing is a chain-termination sequencing method that generally produces a high-quality read for a relatively limited DNA region. It is widely used for targeted sequence confirmation and remains practical for laboratories that test a small number of samples or loci.
In HLA typing, Sanger sequencing is usually not considered completely separate from PCR-SBT. Instead, it is commonly the sequencing component within a PCR-SBT workflow.
Sanger sequencing may be appropriate for:
Confirming a known sequence variant
Characterizing a single HLA locus
Testing a limited number of samples
Verifying PCR products
Supporting development of a new targeted assay
Confirming results produced by another method
Sanger sequencing can provide clear sequence data for the target region and does not require the same level of bioinformatics infrastructure as NGS or TGS. It is also familiar to many molecular laboratories and can be integrated into an existing PCR workflow.
For a single-locus assay, the combination of PCR amplification and Sanger sequencing may offer a reasonable balance between sequence information, equipment requirements, and testing volume.
Sanger sequencing becomes less efficient when many samples, loci, or sequencing directions must be processed. It also may not resolve alleles that differ outside the amplified or read region.
When the assay requires multiple HLA loci, high sample throughput, full-gene characterization, or clear phasing of variants, Sanger-based methods may require too many separate reactions and supplementary tests.
Next-generation sequencing generates many sequence reads in parallel. Depending on the assay design, NGS may use targeted amplicons, hybrid capture, or other enrichment approaches to analyze one or more HLA loci.
NGS can support broader sequence coverage and higher sample throughput than a conventional Sanger workflow. It is therefore often considered when the laboratory needs to process multiple loci or a larger number of samples within one testing program.
NGS may be suitable for:
Multi-locus HLA typing
High-throughput laboratories
Transplantation-related typing
Research panels covering multiple HLA genes
Assays requiring broader sequence coverage
Laboratories that already operate a sequencing and bioinformatics workflow
The main advantage of NGS is scalability. Multiple samples and target regions can be processed in parallel, reducing the need to perform a separate sequencing workflow for every locus and sample.
Targeted NGS panels can also provide more comprehensive information than a limited Sanger assay. Depending on the design, they may improve the ability to detect sequence variation across several exons or other relevant regions.
NGS requires more than a sequencer. The laboratory must manage library preparation, indexing, read-quality assessment, coverage evaluation, contamination monitoring, data storage, software analysis, and allele assignment.
Short-read NGS may also have difficulty resolving variants that are far apart on the same molecule or determining whether variants are in cis or trans. In these cases, the result may remain ambiguous even when overall read depth is high.
Third-generation sequencing, or TGS, reads much longer DNA molecules than conventional short-read sequencing platforms. This longer read length can help connect distant variants, support phasing, and provide a more complete view of an HLA gene.
TGS is particularly relevant when the assay requires high-resolution typing or when short-read and Sanger methods leave unresolved ambiguities.
TGS may be appropriate for:
High-resolution HLA typing
Full-gene or near-full-gene analysis
Phasing of multiple variants
Multi-locus genotyping programs
Complex alleles that are difficult to resolve with shorter reads
Workflows where detailed allele assignment is more important than minimal equipment requirements
Long-read sequencing can provide information about variants located on the same DNA molecule. This may improve allele phasing and help distinguish genotypes that appear similar when only short regions are analyzed.
For comprehensive HLA workflows, TGS can reduce the need to combine multiple independent tests to resolve the same ambiguity. CB-Gene’s HLA matching multi-locus genotyping standards are designed for multi-locus evaluation and are characterized using third-generation sequencing.
TGS may require specialized instruments, library preparation procedures, data-analysis tools, and technical expertise. The laboratory should also consider instrument utilization, consumable cost, throughput, validation requirements, and the level of bioinformatics support available.
A long-read method is not automatically the best choice for every assay. If the assay only needs to detect one known allele, the additional information generated by TGS may not justify the extra workflow complexity.
Method | Typical Information | Best Fit | Main Strength | Main Limitation |
|---|---|---|---|---|
PCR-SSP | Target-specific amplification result | Known allele or allele-group screening | Fast and relatively simple | Limited sequence information |
PCR-SBT | Sequence of selected amplified regions | Focused locus typing | More information than primer-only detection | Ambiguity outside the sequenced region |
Sanger | High-quality sequence from limited targets | Small-scale confirmation and targeted typing | Familiar workflow and clear local sequence data | Limited scalability and coverage |
NGS | Many short reads across selected targets | Multi-locus and higher-throughput assays | Parallel processing and broader coverage | Bioinformatics and phasing challenges |
TGS | Long reads across extended or full-gene regions | High-resolution and complex HLA typing | Supports phasing and long-range resolution | Greater technical and infrastructure requirements |
Start by defining the level of allele information that the assay must report. A screening assay may only need to distinguish a specific allele group, while a transplantation workflow may require detailed allele-level or high-resolution results.
The method should generate enough information to support the intended report. Producing more sequence data than the reporting requirement does not automatically improve the assay.
The related article HLA allele nomenclature and typing resolution explains how reporting depth affects method selection.
A single-locus assay and an 11-locus matching workflow should not be designed around the same technology by default.
PCR-SSP, PCR-SBT, or Sanger may be practical for one or a few loci. NGS or TGS may provide better scalability when multiple class I and class II loci must be evaluated in the same program.
The target loci should also match the selected reference material. A single-locus HLA standard may be appropriate for a focused assay, while a multi-locus HLA genotyping standard may be more relevant to a broad matching workflow.
For a small number of samples, a Sanger-based workflow may be efficient and easy to manage. As sample numbers increase, the laboratory should compare the total workload for PCR setup, sequencing, data analysis, and result reporting.
NGS and TGS can process multiple samples in parallel, but the laboratory must have enough testing volume to use the platform efficiently.
If the assay must determine whether variants occur on the same allele, read length becomes an important consideration.
Short targeted sequencing may not provide sufficient information to phase distant variants. Long-read TGS may be preferable when phasing or full-gene interpretation is central to the assay’s purpose.
Method selection should include the complete workflow, not only the instrument. Consider:
PCR and library preparation equipment
Sequencing instruments
Data-analysis software
HLA allele database access
Bioinformatics expertise
Data storage and reporting systems
Quality control procedures
Technical support and maintenance
A technically powerful method may create operational problems if the laboratory cannot consistently manage its data or interpretation requirements.
The reference material should be characterized at a level that supports the selected assay. Its target loci, allele designation, resolution, sample format, and verification information should be reviewed before testing.
CB-Gene’s HLA reference products use sequence alignment with IMGT/HLA database sequences to confirm genotypes. The company’s single-locus and multi-locus materials can therefore support different stages of focused or comprehensive HLA workflow evaluation.
Our another article, HLA reference standards for assay validation and quality control, provides a broader explanation of how characterized materials are used during assay development and routine QC.
A laboratory only needs to determine whether a defined HLA-B allele or allele group is present. The result is used for a focused research or pharmacogenomic assay.
PCR-SSP may be sufficient when the target is known and a rapid detection result is required. PCR-SBT or Sanger may be added when sequence confirmation is necessary.
A laboratory is optimizing primers and interpretation rules for one HLA locus. It needs to evaluate sequence accuracy but does not need to process dozens of loci or hundreds of samples.
PCR-SBT with Sanger sequencing may provide a practical balance between sequence information and workflow complexity. A characterized single-locus standard can provide an expected genotype for comparison.
A laboratory needs to type several HLA class I and class II loci for donor-recipient matching. It expects a moderate or high sample volume and requires broader sequence coverage.
Targeted NGS may be suitable when the laboratory needs scalable multi-locus testing and has the required bioinformatics resources. A multi-locus reference standard can be used to evaluate the integrated workflow.
A laboratory is dealing with unresolved allele assignments caused by variants distributed across a long HLA gene region. It needs to determine phase and obtain more complete sequence information.
TGS may be the better fit if the laboratory has access to long-read sequencing infrastructure and the assay’s intended use justifies the additional complexity.
The most advanced method is not always the most appropriate method. The assay should generate the information needed for its intended report without adding unnecessary complexity.
PCR-SBT is a workflow, while Sanger is commonly the sequencing technology used within that workflow. Treating them as unrelated options can lead to an unclear method comparison.
A method that is manageable for one locus may become inefficient when applied to a broad multi-locus panel. The total number of PCR reactions, sequencing reactions, data files, and interpretation steps should be considered.
More reads do not automatically resolve phasing or variants that are outside the assay’s target region. Coverage, read length, target design, and allele database interpretation all affect the final result.
NGS and TGS workflows depend heavily on software, databases, quality thresholds, and interpretation rules. These elements should be defined during assay design rather than added after laboratory testing begins.
The right HLA typing method depends on the question the assay must answer. PCR-SSP is well suited to rapid, targeted detection of known alleles or allele groups. PCR-SBT and Sanger sequencing are practical for focused sequence-based typing and confirmation at a limited number of loci. NGS offers greater scalability for multi-locus and higher-throughput workflows, while TGS is valuable when long-range information, phasing, or high-resolution allele assignment is required.
Before choosing a platform, laboratories should define the required resolution, target loci, sample volume, throughput, phasing needs, infrastructure, and reporting requirements. The selected HLA reference standard should match the same analytical scope and provide a reliable expected genotype for method evaluation.
Not exactly. PCR-SBT describes the complete sequence-based typing workflow, while Sanger sequencing is commonly used as the sequencing technology after PCR amplification.
PCR-SSP can be fast for a limited number of known targets because it only detects predefined primer reactions. Actual turnaround time depends on assay design, sample preparation, workflow capacity, and result interpretation.
No. NGS is generally more scalable and can cover more targets in parallel, but Sanger may be more practical for a small number of samples or a focused single-locus assay.
TGS may be more appropriate when the assay requires long reads, phasing, full-gene information, or resolution of ambiguities that remain after short-read sequencing.
Not automatically. The material should match the target loci, allele resolution, sample format, and intended use of the assay. A standard suitable for a focused single-locus workflow may not provide sufficient evidence for a broad multi-locus assay.
NGS or TGS may be suitable depending on the required resolution, read length, throughput, and laboratory infrastructure. TGS may offer additional value when phasing or long-range allele resolution is important.
