Yield of RNA from 200 µL blood was the same (0.1–1 µg approx) regardless of age of blood. The HLA types obtained by sequencing were concordant with those obtained by HLA typing genomic DNA using Luminex. A small number of examples are shown in the Table with the HLA-A, -B, -DR, and -DQ types of three samples. No preferential amplification of alleles, or allele drop-out, was observed. The ambiguities observed are identical to those observed in genomic DNA SBT, as expected.
Ambiguous SBT results are often seen when a “common” allele group is present where the heterozygous sequence of one pair of alleles is identical to at least one other pair of alleles (for HLA-A, -DRB1). In the case of HLA-B the ambiguity arises due to an additional polymorphism found in exon 4, which was not sequenced in the protocol described in this article.
It is debatable whether polymorphisms outside of the peptide binding domains encoded by exon 2 (in HLA-DQB1 and -DRB1) or exons 2 + 3 (HLA-A, -B) are clinically relevant. It is acceptable to report the pair of alleles most common in your population and record the others as “cannot exclude”. Routine HLA-A,B,DR typing by Luminex generally produces a medium resolution result with a number of possible HLA alleles that are abbreviated into a letter code (Table). HLA-DQB1 was typed by PCR-SSP of genomic DNA.
Figure 1 shows a sequencing electropherogram (from SBTengine) covering a short sequence in exon 2 of HLA-DRB1. In this sample exons 1, 2, and 3 were easily sequenced using RNA as template. In genomic DNA this would be quite a feat as exons 1 and 3 in HLA-DRB1 are between 6 and 10 kbp apart, depending on the alleles. In the example shown this allowed resolution of DRB1*14:01/14:54 to DRB1*14:54 and these two alleles only differ in exon 3. To resolve these two alleles one would have to purchase a high-resolution DR14 SSP kit or employ exon 3 amplification prio to sequencing of genomic DNA.
The sequence in Figure 1 clearly shows several heterozygous sequence positions. Among the 64 samples we identified one new HLA-B allele, B*44:59, which had not been identified when this sample was previously HLA typed using genomic DNA. This unique heterozygous position defining B*44:59 (in combination with the other allele) is shown in Figure 2.