High resolution HLA typing is essential for providing HLA matching in solid organ and bone marrow transplantation. Massively parallel Next Generation Sequencing is the current gold standard for high resolution typing however bottlenecks in preparing the DNA library for sequencing can impede turnaround times, particularly for high-throughput donor registry typing in the absence of automation.
A Short Protocol was developed for NGS assays, aiming to address library preparation bottlenecks by streamlining the classic NGS assay for registry workflows. Time-consuming steps such as amplicon quantitation, normalisation and clean-up have been reduced, while the time taken to pool samples into a single tube has been shortened, greatly simplifying manual size selection steps. The feasibility study included the development of a 384-index kit which allows for pooling and simultaneous sequencing of greater sample numbers compared to the 96 barcodes.
An initial trial run of the NGS Short Protocol, using representative NGS assay AllType, demonstrated successful processing of a n=192 library in similar time frames to a n=48 classic NGS run – representing a 4-fold increase in throughput. When sequenced on an Illumina MiniSeq with a high output reagent kit, the run was broadly comparable to classic NGS in terms of data quality, however lower total reads per barcode were a natural trade-off of higher sample count versus sequencing yield. With the removal of processing bottlenecks, data analysis practices and settings may also require optimisation to improve the sequencing to result turnaround for high throughput runs.