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SSO, SSP, SBT and NGS HLA typing compared

Updated 2026-10-30 · allele examples checked against IPD-IMGT/HLA 3.66.0 on 2026-10-30

SSO, SSP, SBT and NGS are the four main families of DNA-based HLA typing. SSO and SSP detect known sequence motifs with probes or primers; SBT reads the sequence of targeted exons directly; NGS sequences single DNA molecules, usually over longer regions, which lets it assign variants to one copy of the gene.

The practical difference for anyone handling the results is what each method leaves unresolved: which alleles it cannot tell apart, and whether it can say which variants sit on the same chromosome. That determines how precise the reported names can honestly be. This guide summarises two peer-reviewed reviews, a 2024 review of typing techniques by Geo and colleagues and a 2013 workshop review of NGS typing by De Santis and colleagues, and does not compare commercial products.

The four methods side by side

"Intermediate resolution" is the review's wording. Formal resolution terms have their own published definitions, from a 2011 harmonisation working group; check those and your protocol rather than relying on any one review's labels.

SSO: probes for known motifs

In SSO typing, the HLA gene is amplified and the product is hybridised to a panel of oligonucleotide probes, each matching a known sequence motif. Which probes bind is then interpreted against the motifs of named alleles.

Geo and colleagues describe SSO and SSP as methods that "identify specific, previously known sequence motifs". Two limits follow:

• Novel alleles. Both methods are "limited in their ability to screen only predetermined sets of known polymorphisms, potentially leading to mistyping if a novel allele is present". A new allele can produce a probe pattern that matches a known one.
• Phase. SSO "does not distinguish between cis and trans polymorphisms". In a heterozygous sample, the probes say which motifs are present, not which copy carries them.

SSP: primers that amplify or do not

In SSP typing, a set of PCR reactions each uses primers that amplify only when specific sequences are present, and the reactions are read as the presence or absence of a band, alongside an internal control. The review describes SSP as distinguishing alleles "based on the presence or absence of specific bands" notes that each reaction runs with a control, and says the technique "allows some phasing by using both forward and reverse primers".

Its resolution depends entirely on the primer set. Like SSO, it can only detect polymorphisms someone designed a reaction for.

SBT: reading the targeted exons

Sanger sequence-based typing amplifies the polymorphic exons of a locus "using universal oligonucleotide primers targeting conserved sections of a locus", in the 2024 review's words, and sequences the product. Both copies of the gene are sequenced together, so a heterozygous position appears as a mixed signal.

That gives SBT its two characteristic ambiguities:

1. Untargeted regions. "Typing ambiguities can occur when two or more alleles share the same sequences in the targeted exons but differ in the untargeted exons." The 2013 review notes that only exons 2 and 3 for class I, and exon 2 for class II, the regions encoding the peptide-binding site, were routinely assessed.
2. Phase. Sanger sequencing of both copies at once cannot tell whether two heterozygous positions are on the same chromosome or on different ones. The 2013 review attributes ambiguous combinations to "cis-trans ambiguities or of particular allele combinations being identical over the regions commonly analysed".

The WMDA README in the IPD-IMGT/HLA repository gives the same reason for publishing the G group file: most SBT uses exons 2 and 3 for class I and exon 2 for class II, and the heterozygous nature of the analysis means many pairs of alleles can give an ambiguous result. G groups, alleles identical over those exons, are the nomenclature's way of reporting the first kind of ambiguity.

The 2024 review adds one consequence that matters for data: focusing on the antigen recognition site hinders "detection of null alleles carrying mutations outside the ARS". A null allele whose defect lies in another exon can be reported as its expressed relative. The null alleles guide covers how often null alleles share a G group with expressed ones.

NGS: single molecules and longer regions

The 2013 workshop review summarises what NGS changed: "the combination of clonal amplification, which provides phase information, and the ability to sequence larger regions of genes, including introns". Each read comes from one DNA molecule, so variants within the same read are known to be on the same copy. The 2024 review says NGS permits "the direct determination of phase for heterozygous nucleotides" and can "enable the sequencing of entire HLA genes", including non-coding regions.

That attacks both SBT ambiguities. It does not remove them completely. The 2024 review notes that with short reads "the inability to phase distant polymorphic regions leads to ambiguity", and alleles that differ only outside the sequenced region remain indistinguishable. A result can still be a list.

What this means for the names in a report

The method sets a ceiling on what the reported names can mean:

• A probe or primer result is a pattern match against known alleles. The names it reports are the known alleles consistent with the pattern, as interpreted against whichever allele list the interpretation used.
• An exon-based SBT result is resolved over the exons sequenced. A G group name, such as A*02:01:01G, says exactly that: one of the alleles identical over exons 2 and 3. In IPD-IMGT/HLA release 3.66.0 that group has 554 members, including null alleles such as A*02:43N.
• An NGS result can reach full-length names, such as A*02:01:01:01, where the sequenced region and phasing support it, and should still state any residual ambiguity.

Whichever method produced it, store three things together: the reported string, the method or region examined, and the IPD-IMGT/HLA release the names refer to. The typing resolution guide explains why the length of a name is not the same as the resolution of the typing, and the ambiguity guide shows how to keep allele, genotype and phase ambiguity apart in GL String.

Frequently asked questions

What is the difference between SSO and SSP HLA typing?

SSO tests amplified DNA against probes for known motifs; SSP uses primer pairs that amplify only when specific sequences are present, read as bands. Both detect only polymorphisms that were designed into the assay.

Why does Sanger SBT give ambiguous HLA results?

Because it sequences both copies at once and usually only the exons encoding the peptide-binding domains. It cannot always assign heterozygous positions to one copy, and it cannot see differences in exons it did not sequence.

Does NGS HLA typing eliminate ambiguity?

It reduces it, because clonal reads carry phase and more of the gene can be sequenced. Published reviews note that short reads can still leave distant positions unphased.

Can an exon-based typing method miss a null allele?

Yes. The 2024 review notes that focusing on the antigen recognition site hinders detection of null alleles whose mutations lie outside it.