Massively parallel and multiplex blood group genotyping using next-generation-sequencing.

OBJECTIVES: Thirty-six blood group systems are listed by the International Society of Blood Transfusion, containing almost 350 antigens. Most of these result from a single nucleotide polymorphism (SNP). Serology is the standard method for blood group typing. However, this technique has some limitations and cannot respond to the growing demand of blood product typing for a large number of antigens. Here we describe a blood group genotyping assay directly from whole blood samples using Next-Generation Sequencing (NGS), allowing the simultaneous identification of 15 SNPs associated with the blood group systems of 95 patients in a single run.

DESIGN AND METHOD: After an automated DNA extraction, targets are amplified by multiplex polymerase chain reaction (PCRm). Two panels addressing 9 groups have been developed (MNS, Lutheran, Kell, Duffy, Kidd, Diego, Yt, Dombrock, and Colton), one for 8 SNPs, the other for 7 SNPs. For each sample, both panels corresponding to 14 amplicons (1 amplicon containing 2 SNPs) are pooled. Then a dual-indexed library is generated from each pool by linking Illumina adaptors directly onto amplicons, followed by sequencing using the MiSeq platform (Illumina).

RESULTS: In a single experiment, 95 blood donor samples have been sequenced for the genes of interest. Among the 1425 targeted single nucleotide polymorphisms, 1420 were identified by sequencing, reflecting a coverage of 99.65%. The obtained data shows a good correlation (99% for all SNPs) with other blood group typing methods. Depending on the allele pairs analyzed, correlations vary between 97.12 and 100%.

CONCLUSION: Next-Generation sequencing would supplement serological and molecular techniques and, in the near future, could replace it with complete and fast results acquisition for pre-screening and identification of rare blood bags.