Aim: DNA extracted from peripheral blood has proven to be a reliable source of genetic material for NGS high resolution HLA typing due to high quality and yields. Finding an alternative source such as buccal or saliva DNA that is less invasive, fast, and cost effective would be extremely beneficial for clinical laboratories. Hybrid Capture is the preferred NGS typing method for DNA samples with variable quality and yields that often underperform with PCR-based assays. In this study, we compared high resolution typing results for buccal and saliva DNA samples extracted from the same donor and processed with a Hybrid Capture NGS kit that enables the enrichment of up to 19 Loci with three probe modules. We compared QC metrics for sample types and assessed the overall data of the Hybrid Capture NGS kit.
Methods: Buccal swab and saliva samples were collected in tandem from 24 donors. Genomic DNA was extracted using the MagMAX™ DNA Multi-Sample Ultra 2.0 kit and the KingFisher™ Flex automation system. All extracted DNA samples were evaluated by Qubit and TapeStation for quality checks and subsequently processed with a 19 Loci Hybrid Capture NGS kit following the manufacturer protocol. Resulting NGS libraries were sequenced on the MiSeq i100 instrument using the 25M Flow Cell and 2 x 150bp read length. Fastq files were analyzed on the TypeStream™ Visual Software version 3.2 for genotypic assignments.
Results: DNA concentrations from buccal and saliva samples were in the 5-200ng/uL concentration range and fragment peaks were ≥ 7.5Kb with variable levels of smaller fragments. Mixed sample type libraries processed with the 19 Loci Hybrid Capture NGS kit yielded high quality reads with % Q30 > 90% and > 50 M paired end reads in less than 8 hours. Typing results between sample types were equivalent at Field-3 for Classical and Non-Classical HLA genes and Field-2 for MIC genes. No differences in the ABO phenotypes were found. The percentage of reads that mapped to 19 Loci was > 70% and coverage across all exon regions was > 100 for all loci tested.
Conclusion: Paired DNA samples extracted from buccal swabs and saliva showed equivalent results and robust data using a Hybrid Capture NGS kit for 19 Loci.
