Aim: Next generation sequencing (NGS) based HLA-typing is a powerful tool that offers unparalleled high quality typing resolution. One of the main challenges with NGS assays are long and sensitive workflows, often spanning multiple days with many steps that require careful attention. The goal of this project is to combine the power of a highly multiplexed HLA-typing NGS assay with the precision and flexibility of the HLA PRO Instrument, a general-purpose liquid handling pipettor to automate Illumina library preparation for NGS HLA-typing assays.
Method: We developed amplicon purification and library preparation scripts for the HLA PRO instrument and tested the performance of these scripts on multiple instruments, with various number of samples being processed, and also investigated reagent stability (over time and upon freezing/defrosting). The performance of the HLA PRO instrument and script workflows were assessed at multiple stages– amplicons yield, NGS library quality, and finally HLA locus coverage and typing accuracy.
Results: The HLA PRO instrument demonstrated robust performance for NGS library preparation across many conditions– various throughputs, multiple kit uses, and different instruments. Amplicon purification yielded quality material for subsequent library preparation steps and was completed in less than 1.5 hours for up to 96 samples. The automated library preparation generated high quality libraries with concentrations greater than 5 ng/µL. Sequencing depth was greater than 200 for key exons, which along with exon 1 coverage to resolve additional ambiguities resulted in 11 HLA loci typing that was >99% concordant to the third field across all samples tested. We found that multiple freeze thaws for library preparation reagents had no impact on library quality or sequencing results, indicating that this process can be adapted for various use configurations.
Conclusion: The HLA PRO Instrument and workflow scripts automate multiplexed-amplicon purification and library preparation to deliver high quality libraries for 11 HLA loci and exon 1 in under 8 hours. This flexible platform allows for library preparation for as few as 8 or as many as 96 samples, and creates libraries that are fully compatible with Illumina sequencers. This workflow dramatically reduces hands on time for NGS library preparation, while consistently generating high-quality and high-resolution HLA typing data.
