Haplotype sequence collection of ABO blood group alleles by long-read sequencing reveals putative A1-diagnostic variants

Abstract

In the era of blood group genomics, reference collections of complete and fully resolved blood group gene alleles have gained high importance. For most blood groups, however, such collections are currently lacking, as resolving full-length gene sequences as haplotypes (ie, separated maternal/paternal origin) remains exceedingly difficult with both Sanger and short-read next-generation sequencing. Using the latest third-generation long-read sequencing, we generated a collection of fully resolved sequences for all 6 main ABO allele groups: ABO∗A1/A2/B/O.01.01/O.01.02/O.02. We selected 77 samples from an ABO genotype data set (n = 25 200) of serologically typed Swiss blood donors. The entire ABO gene was amplified in 2 overlapping long-range polymerase chain reactions (covering ∼23.6 kb) and sequenced by long-read Oxford Nanopore sequencing. For quality validation, 2 samples per ABO group were resequenced using Illumina and Pacific Biosciences technology. All 154 full-length ABO sequences were resolved as haplotypes. We observed novel, distinct sequence patterns for each ABO group. Most genetic diversity was found between, not within, ABO groups. Phylogenetic tree and haplotype network analyses highlighted distinct clades of each ABO group. Strikingly, our data uncovered 4 genetic variants putatively specific for ABO∗A1, for which direct diagnostic targets are currently lacking. We validated A1-diagnostic potential using whole-genome data (n = 4872) of a multiethnic cohort. Overall, our sequencing strategy proved powerful for producing high-quality ABO haplotypes and holds promise for generating similar collections for other blood groups. The publicly available collection of 154 haplotypes will serve as a valuable resource for molecular analyses of ABO, as well as studies about the function and evolutionary history of ABO.

Graphical abstract

Figure 1.

Genetic structure of the ABO gene locus, including long-range PCR (LR-PCR) amplicon locations and positions of the 4 putative ABO∗A1-diagnostic variants. The coordinates above the gene and next to the PCR primers correspond to the reference sequence NG_006669.2 (LRG_792). Chromosomal coordinates of the first base pair of ABO reference gene on the current human genome reference (GRCh38.p13) is provided underneath the gene. Exons 1 to 7 are represented by black arrows, and the coding DNA sequence (CDS; reference transcript NM_020469.3) in red. The locations of the 2 overlapping LR-PCR amplicons (LR1 and LR2) used to amplify the ABO gene are highlighted by the striped area between both amplicons. The positions of the 4 putative ABO∗A1-specific variants found in this study are indicated by their respective rs numbers. For graphical clearness, the ABO gene is shown as reverse complement.

Figure 2.

Alignment of a random subset of haplotype sequences highlighting distinct sequence patterns of ABO groups. For each ABO group, 6 haplotype sequences were randomly picked. For the subgroup ABO∗O.01.01, we show 6 sequences for both subgroups (g1 and g2) observed in the phylogenetic tree (Figure 3) and haplotype network (Figure 4). Black bars on the haplotype sequences highlight positions that are different to the ABO reference sequence (NG_006669.2). The identity graph (Identity) above the sequences indicates the mean pairwise identity over all sequence pairs by gene position; green represents 100% identity, orange identity between 30% and 99%, and red identity <30%. The ABO gene structure is provided at the top of the graph for orientation purpose.

Figure 3.

Maximum-likelihood phylogenetic tree based on the entire ABO gene locus. All ABO groups form distinct evolutionary clades with an additional split of ABO∗O.01.01 into 2 subgroups (g1 and g2). Bootstrap support is provided for main branching points. The tree was rooted with central chimpanzee sequence (not shown).

Figure 4.

Median-joining haplotype network inferred from all 154 ABO sequences. Phylogenetic network showing the evolutionary relationships among ABO haplotypes. Each circle represents a unique haplotype with the size being proportional to the number of sequences represented. Haplotype circles are colored according to the ABO allele groups; black dots represent missing intermediate haplotypes (ie, unsampled, likely ancient haplotypes). Mutational steps between haplotypes are displayed as hatch marks along the connection lines. The length of the connection lines is not scaled by phylogenetic distance.