Aim: Targeted amplification of HLA by PCR combined with Next Generation Sequencing (NGS) is widely implemented in transplant diagnostics to characterize up to 11 HLA loci in high (third field) resolution. Blood type (ABO) is also a critical piece of information that informs transplant decision-making and organ selection. Current methods for ABO phenotyping are largely performed using whole blood serological testing, but these tests can be ambiguous or inaccurate. Therefore, an accurate and efficient method to provide genetic resolution of ABO is needed.
Method: An ABO-specific primer mix was designed to be combined with an 11 loci HLA NGS primer mix for co-amplification in the same PCR reaction. Twenty-four well characterized samples were amplified with the combined ABO and HLA primer mixes. The amplicons were then processed using the standard HLA NGS workflow and sequenced on an Illumina’s NextSeq 1000.
Results: Analysis of sequencing reads containing ABO and HLA requires a modified algorithm in addition to catalog and reference files for ABO. HLA genotyping is unaffected by addition of ABO as the primer design is balanced to not impact the number of reads required for HLA. Results from 24 samples tested provided 100% HLA concordance. ABO results can be viewed as either phenotype or genotype (including ABO subtype), with the ability to toggle between both available in the software.
Conclusion: An ABO primer mix can be combined with 11 loci HLA primers for PCR amplification, NGS library preparation, and sequencing. The downstream analysis requires software that is calibrated for ABO, but there is no impact to HLA genotype results while there is significant added benefit to genotype level resolution of ABO in the same sequencing run. Furthermore, co-amplification of HLA and ABO reduces hands on time and cost.
