ACKR1 Alleles at 5.6 kb in a Well-Characterized Renewable US Food and Drug Administration (FDA) Reference Panel for Standardization of Blood Group Genotyping

Abstract

The glycoprotein encoded by the ACKR1 gene expresses the Duffy blood group antigens and is a receptor for malaria parasites. We recently described 18 long-range ACKR1 alleles in an autochthonous population of a malaria endemic region. Extending this work, we sequenced the gene in a 53-sample repository established by the US Food and Drug Administration (FDA) as reference reagents for blood group genotyping. The FDA samples have been characterized for 19 genes; however, long-range haplotype information for these genes, including ACKR1, was lacking. We used a hybrid approach, novel for this type of gene, to characterize ACKR1 by combining two next-generation sequencing technologies, the short-read massively parallel sequencing and the long-read nanopore sequencing. The expedient integration of data from both next-generation sequencing systems were necessary and sufficient to allow determination of all 25 long-range ACKR1 alleles found in the 53 samples accurately. All 25 alleles identified in our current FDA cohort were novel and, unexpectedly, none had been observed among the 18 alleles in our previous study. The alleles will be useful for validation, calibration, and proficiency testing of red cell genotyping. The lack of any overlap between the ACKR1 alleles in the two studies documents differences in mutation rate and recombination frequency among populations. The exact haplotype and their interethnic or interpopulation dissimilarities can influence disease susceptibility and therapy.

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

“…Currently, reports of its application are still limited to the very short ACKR1 gene encoding the Duffy blood group. 1 , 23 …”

“…Currently, reports of its application are still limited to the very short ACKR1 gene encoding the Duffy blood group. 1,23 Even for ABO, the first discovered, 24 and clinically most important blood group system, 25 haplotype sequence collections are still lacking. The most comprehensive collection of ABO sequences by short-read sequencing only covers a small exonic part of the gene.…”

CD59 gene: 143 haplotypes of 22,718 nucleotides length by computational phasing in 113 individuals from different ethnicities

“…To obtain high‐quality reference sequences, third‐generation sequencing methods and novel computational techniques are essential and currently being used such as Pacific Biosciences (PacBio) 51 and Oxford Nanopore Technologies (ONT) 52 . We have previously integrated data from two next‐generation sequencing technologies, the short‐read massively parallel sequencing and the long‐read nanopore sequencing, to reliably allow determination of ACKR1 haplotypes without ambiguity 25 . Later, the T2T consortium used a similar long‐read sequencing technology to construct a complete human reference genome 12 .…”

“…The Center for Biologics Evaluation and Research of the FDA provided genomic DNA from 53 volunteer blood donor samples. The FDA cohort consisted of 38 Caucasian and 13 African American individuals, and one individual each from Hispanic and American Indian ethnicity 25,26 . All individuals have been genotyped for five blood group systems 27 while 18 individuals have been red cell genotyped for another 12 blood group systems 26 .…”

“…The FDA cohort consisted of 38 Caucasian and 13 African American individuals, and one individual each from Hispanic and American Indian ethnicity. 25,26 All individuals have been genotyped for five blood group systems 27 while 18 individuals have been red cell genotyped for another 12 blood group systems. 26 Blood samples from 60 other volunteer blood donors were collected from a distinct East African region as published previously, transported to NIH under the NCT01282021 protocol, and genomic DNA was extracted.…”