Evaluation of targeted exome sequencing for 28 protein-based blood group systems, including the homologous gene systems, for blood group genotyping.
Blood group single nucleotide polymorphism genotyping probes for a limited range of polymorphisms. This study investigated whether massively parallel sequencing (also known as next-generation sequencing), with a targeted exome strategy, provides an extended blood group genotype and the extent to which massively parallel sequencing correctly genotypes in homologous gene systems, such as RH and MNS.



Donor samples (n = 28) that were extensively phenotyped and genotyped using single nucleotide polymorphism typing, were analyzed using the TruSight One Sequencing Panel and MiSeq platform. Genes for 28 protein-based blood group systems, GATA1, and KLF1 were analyzed. Copy number variation analysis was used to characterize complex structural variants in the GYPC and RH systems.



The average sequencing depth per target region was 66.2 ± 39.8. Each sample harbored on average 43 ± 9 variants, of which 10 ± 3 were used for genotyping. For the 28 samples, massively parallel sequencing variant sequences correctly matched expected sequences based on single nucleotide polymorphism genotyping data. Copy number variation analysis defined the Rh C/c alleles and complex RHD hybrids. Hybrid RHD*D-CE-D variants were correctly identified, but copy number variation analysis did not confidently distinguish between D and CE exon deletion versus rearrangement.



The targeted exome sequencing strategy employed extended the range of blood group genotypes detected compared with single nucleotide polymorphism typing. This single-test format included detection of complex MNS hybrid cases and, with copy number variation analysis, defined RH hybrid genes along with the RHCE*C allele hitherto difficult to resolve by variant detection. The approach is economical compared with whole-genome sequencing and is suitable for a red blood cell reference laboratory setting.
Research and Development.
Elizna M
EM
Schoeman
Research and Development.
Genghis H
0000-0001-8568-0604
GH
Lopez
Research and Development.
Eunike C
EC
McGowan
Research and Development.
Glenda M
GM
Millard
Research and Development.
Helen
H
O'Brien
Research and Development.
Eileen V
EV
Roulis
Red Cell Reference Laboratory, Australian Red Cross Blood Service, Brisbane, Queensland, Australia.
Yew-Wah
YW
Liew
Red Cell Reference Laboratory, Australian Red Cross Blood Service, Brisbane, Queensland, Australia.
Jacqueline R
JR
Martin
Red Cell Reference Laboratory, Australian Red Cross Blood Service, Brisbane, Queensland, Australia.
Kelli A
KA
McGrath
Red Cell Reference Laboratory, Australian Red Cross Blood Service, Brisbane, Queensland, Australia.
Tanya
T
Powley
Research and Development.
Robert L
RL
Flower
Research and Development.
Catherine A
CA
Hyland
D005260:Female; D015894:Genome, Human; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D008297:Male; D020641:Polymorphism, Single Nucleotide; D012204:Rh-Hr Blood-Group System / Q000235:genetics*
C066468:RHCE protein, human; D012204:Rh-Hr Blood-Group System