Aim: HLA typing is routinely performed on DNA obtained from peripheral blood, but buccal swab DNA can be advantageous due to the relative ease of collection and transport to the laboratory. However, buccal DNA tends to have increased allele dropout or total typing failures compared to PB DNA due to poorer DNA quality, notably a higher degree of fragmentation. Hybrid capture method may be more tolerant of fragmented samples as compared to Long Range PCR approaches. In this study, paired buccal and PB DNA collected from the same individuals were selected to evaluate the performance of buccal swab DNA for HLA typing using hybrid capture technology. In addition, varying degrees of fragmentation were assessed to determine the effect on hybrid capture assay performance and Long-Range PCR (LR-PCR), respectively.
Method: Twenty-six buccal swabs were collected from twenty-three individuals and were extracted using either Qiagen Blood Mini kit (18) or Maxwell Promega RSC Genomic kit (8). Seven of the individuals also submitted PB samples and were extracted with the Qiagen Blood Mini kit. Severely fragmented DNA was created with NEBNext® dsDNA fragmentase per kit instructions, with varying incubation times. DNA was assessed by Nanodrop™ Lite, Qubit 4 fluorometer, and Tapestation 4150. All samples were prepared with AlloSeq Tx17™ and sequenced utilizing a Miseq with a micro flow cell. LR-PCR was performed using in-house primers.
Results: DNA fragmentation varied widely between individual buccal swab samples, which resulted in lower Tx17 library concentration vs. PB samples. Regardless, HLA typing results were 100% concordant to three fields between individuals that supplied both buccal and PB samples. Severely fragmented DNA samples which also had reduced library concentrations, but typing was successful and 100% concordant. Notably, LR-PCR showed a degree of failure with the severely fragmented DNA samples.
Conclusion: Buccal swabs are an acceptable sample source for HLA typing with AlloSeq Tx17™ hybrid capture technology. DNA fragmentation varies between individuals which may explain inconsistent success seen with LR-PCR technologies. The increased fragmentation seen with buccal DNA vs PB DNA will decrease the overall library concentration but may be accommodated through compensatory pooling strategies.
