Aim: Sequencing the human leukocyte antigen (HLA) region offers essential information for transplant diagnosis. HLA typing via next-generation sequencing (NGS) offers remarkable flexibility and achieves high resolution. Some laboratories may encounter challenges in adopting NGS because of its heightened complexity and demanding labor needs. To streamline this procedure and alleviate labor demands, we have automated the workflow for an 11 loci NGS library preparation assay on HLA PRO, built upon the framework of the Tecan Fluent pipettor instrument. The HLA PRO can fully automate library preparation for 8 to 48 samples with unique barcodes and common sequences for subsequent amplification at the ends of DNA fragments to match the Ion Torrent system.
Method: Two scripts were tested: one for the automated purification of amplicons containing the targeted HLA genes via PCR, and the other for fully automating the processes of amplicon dilution, sample barcoding, pooling, universal barcoding, amplification, and final library purification steps.
Results: To evaluate the efficacy of the automated amplicon purification process, identical amplicons were pooled and underwent purification using the HLA PRO system. Quantitative analysis has shown that the system effectively purified the amplicons, yielding high concentrations with minimal variability. The library preparation script demonstrated robust and reproducible results. The HLA PRO system carried out the library preparation steps, resulting in sequencer-ready fragments. The sequencing libraries exhibited good yield and contained fragments of appropriate sizes for the Ion Torrent NGS system. The sequencing coverage was robust, ensuring a minimum read depth of 200 across key exon regions. HLA-typing of libraries prepared on the HLA PRO were concordant with those prepared manually. The entire library preparation procedure was typically 6.5 hours, with hands-on time minimized to under 0.5 hours.
Conclusion: The HLA PRO can generate a multiplexed HLA library comprised of sequencer-ready fragments derived from amplified genomic DNAs. The automated HLA PRO significantly decreases the hands-on time required to prepare the library for sequencing on the Ion Torrent system.
