Targeted exome sequencing designed for blood group, platelet, and neutrophil antigen investigations: Proof‐of‐principle study for a customized single‐test system

Abstract

Background Immunohematology reference laboratories provide red blood cell (RBC), platelet (PLT), and neutrophil typing to resolve complex cases, using serology and commercial DNA tests that define clinically important antigens. Broad‐range exome sequencing panels that include blood group targets provide accurate blood group antigen predictions beyond those defined by serology and commercial typing systems and identify rare and novel variants. The aim of this study was to design and assess a panel for targeted exome sequencing of RBC, PLT, and neutrophil antigen–associated genes to provide a comprehensive profile in a single test, excluding unrelated gene targets. Study Design and Methods An overlapping probe panel was designed for the coding regions of 64 genes and loci involved in gene expression. Sequencing was performed on 34 RBC and 17 PLT/neutrophil reference samples. Variant call outputs were analyzed using software to predict star allele diplotypes. Results were compared with serology and previous sequence genotyping data. Results Average coverage exceeded 250×, with more than 94% of targets at Q30 quality or greater. Increased coverage revealed a variant in the Scianna system that was previously undetected. The software correctly predicted allele diplotypes for 99.5% of RBC blood groups tested and 100% of PLT and HNA antigens excepting HNA‐2. Optimal throughput was 12 to 14 samples per run. Conclusion This single‐test system demonstrates high coverage and quality, allowing for the detection of previously overlooked variants and increased sample throughput. This system has the potential to integrate genomic testing across laboratories within hematologic reference settings.

RBCeq: An Integrated Bioinformatics Algorithm Designed to Improve Blood Type Compatibility Testing

“…245 targeted blood group exome sequencing samples with serological phenotypes for ABO, D, C, c, E, e, K and k blood groups(30). This dataset was important to validate the accuracy of the algorithm with established methods currently used to determine ABO, D, C, c, E, e, K, k phenotypes.…”

“…Proof of Principle Algorithm Development dataset: Initial development was undertaken using 13 in-house targeted exome sequencing (TES) samples for which serological data for 14 blood groups (+ 4 samples with SNParray) were available with the remaining 22 blood groups were predicted (27), provided in the supplementary table 3. In addition 45 previously published complex blood group serology cases with TES, from the Australian Red Cross Lifeblood Red Cell Reference Laboratory with manually predicted blood groups (Supplementary Table 4) (17,27,30) were analysed.…”

“…South East Queensland Indigenous TES data: 245 targeted blood group exome sequencing samples with serological phenotypes for ABO, D, C, c, E, e, K and k blood groups (30) 2504 WGS bam files were accessed through the 1000G project FTP server.…”

RBCeq: A robust and scalable algorithm for accurate genetic blood typing

“…(Supplementary Table 4). 17,18,30 South East Queensland Indigenous TES data: This dataset comprised 244 targeted blood group exome sequencing samples with serologically validated phenotypes for ABO, D, C, c, E, e, K and k blood groups. 3 MedSeq project: 110 whole-genome sequencing samples (30X) from the MedSeq Project randomized controlled trial (accession number phs000958) were accessed through dbGaP authorized access.…”

Using Whole Genome Sequencing to Characterize Clinically Significant Blood Groups Among Healthy Older Australians

“…[20][21][22][23][24] NGS approaches including targeted exome sequencing (TES), whole exome sequencing (WES), and whole genome sequencing (WGS) have the potential to provide a new basis of pre-transfusion testing by facilitating the accurate characterization of an individual's complete blood group profile, supporting precision-based medicine. 25,26 Curated and detailed DNA-phenotype-annotated databases storing blood group allele and antigen data has been developed by ISBT, providing tools for blood type calling from genetic data. As NGS is increasingly employed in immunohematology, Erythrogene, a customdesigned blood group allele database associated with the 1000 Genomes (1KGP3) project has been developed and applied.…”