A novel multiplexed 11 locus HLA full gene amplification assay using next generation sequencing

Area of Science:

• Immunogenetics
• Molecular Biology
• Genomic Medicine

Background:

• Hematopoietic stem cell transplantation (HSCT) outcomes are improving due to advancements in Human Leukocyte Antigen (HLA) typing.
• Unambiguous HLA typing remains a significant challenge in transplantation medicine.
• Next-generation sequencing (NGS) offers potential to resolve HLA typing ambiguities and streamline workflows.

Purpose of the Study:

• To develop a multiplexed, full-gene PCR assay for 11 HLA loci.
• To enable comprehensive HLA typing compatible with any NGS platform.
• To provide additional genetic information beyond traditionally sequenced regions.

Main Methods:

• Amplification of 11 HLA loci (HLA-A, -B, -C, DRB1, DRB3/4/5, DQB1, DQA1, DPB1, DPA1) in four multiplexed PCR reactions.
• Evaluation of the assay using a DNA reference panel (47 samples) on the Ion Torrent sequencing platform.
• Validation of specificity and sensitivity on an additional 158 Caucasian control samples.

Main Results:

• Full gene sequences (start to stop codons, including UTRs) were obtained for all 11 HLA loci.
• Complete gene coverage and sufficient read-depth were achieved for 3619 alleles.
• 100% concordance was observed for full gene resolution (Class I) and exon resolution (Class II) compared to existing NGS and Sanger methods.

Conclusions:

• The novel multiplexed PCR approach facilitates simple and rapid full-gene HLA typing.
• This method generates a substantial amount of genetic information, enhancing HLA typing capabilities.
• The assay is suitable for broad application across different NGS platforms, supporting improved transplantation strategies.