Single molecule real‐time DNA sequencing of the full HLA‐E gene for 212 reference cell lines

Abstract

We have developed a genotyping assay that produces fully phased, unambiguous HLA-E genotyping using Pacific Biosciences' single molecule real-time DNA sequencing. In total 212 cell lines were genotyped, including the panel of 107 established at the 10th International Histocompatibility Workshop. Our results matched the previously known HLA-E genotype in 94 (44.3%) cell lines, in all cases either improving or equalling previous genotyping resolution. Three (1.4%) cells had discrepant HLA-E genotyping data and 115 (54.2%) had no previous HLA-E data. The HLA-E genotypes for four (1.9%) cell lines resulted in a change of zygosity by identifying two distinct haplotypes. We discovered eight novel HLA-E alleles, extended the known reference sequence of seven and confirmed the existence of a further 10.

86 novel HLA‐E alleles discovered through full‐gene sequencing of 6227 hematopoietic cell transplant patients and unrelated donors

“…Moreover, this 28 bp sequence has also been observed repeated four times in another HLA-E allele (HLA-E*01:03:02:07) as described previously. 11 We found the combined frequency in this cohort of HLA-E*01:01 and HLA-E*01:03 alleles was 97.5%, which is in line with the previously published data. 7,8,16 It should be noted however, that as this cohort is not a random population sample we acknowledge that allele frequencies presented from this cohort do not necessarily represent the true frequencies in populations.…”

“…The historical dogma in HLA of only sequencing exons 2 and 3 of class I genes has likely compounded the apparent lack of genetic diversity in HLA‐ E, influencing the most published studies to only sequence exons 2 and 3 and therefore only obtaining second field resolution HLA‐E genotypes. Although exons 2 and 3 are translated into the antigen recognition domain which forms direct molecular interactions with the bound peptide, there is likely to be further genetic polymorphism outside these regions that could be important in immune system related studies, as has been found by ourselves and others with the classical HLA genes 11–13 …”

“…Although exons 2 and 3 are translated into the antigen recognition domain which forms direct molecular interactions with the bound peptide, there is likely to be further genetic polymorphism outside these regions that could be important in immune system related studies, as has been found by ourselves and others with the classical HLA genes. [11][12][13] Here, we present data from our full-gene sequencing of HLA-E using Pacific Biosciences' single-molecule realtime (SMRT) DNA sequencing technology, providing fully phased, unambiguous genotypes for 6227 samples. In total, DNA from 2468 patients undergoing an allogeneic HCT and 3759 unrelated donors requested for verification typing as potential donors, were genotyped for HLA-E. PCR amplification of HLA-E was performed with Takara La Taq DNA polymerase using standard PCR cycling conditions and in-house designed primers located within the 5 0 and 3 0 untranslated regions (UTR) that captured all currently known HLA-E polymorphisms, as described previously.…”

“…In total, DNA from 2468 patients undergoing an allogeneic HCT and 3759 unrelated donors requested for verification typing as potential donors, were genotyped for HLA-E. PCR amplification of HLA-E was performed with Takara La Taq DNA polymerase using standard PCR cycling conditions and in-house designed primers located within the 5 0 and 3 0 untranslated regions (UTR) that captured all currently known HLA-E polymorphisms, as described previously. 11 Primer sequences can be made available upon reasonable request. Libraries were sequenced by either a Pacific Biosciences RS II or Sequel machine and genotyping performed by our in-house HLA genotyping pipeline.…”

HLA Class I Molecules as Immune Checkpoints for NK Cell Alloreactivity and Anti-Viral Immunity in Kidney Transplantation

“…In this latter study HLA-E was typed for over 2.5 million potential stem cell donors worldwide and although only a limited 535 bp amplicon (including last part of exon 2, intron 2 and first part of exon 3) was sequenced, it has caused an explosion of new HLA-E alleles (209). Also full length sequencing of HLA-E was developed using both Sanger sequencing (210,211) and NGS (212)(213)(214)(215)(216). These full length strategies have also revealed new alleles, including alleles with polymorphism present outside of the peptide binding groove.…”