2.7 million samples genotyped for HLA by next generation sequencing: lessons learned

Area of Science:

• Immunogenetics
• Molecular Biology
• Bioinformatics

Background:

• Next Generation Sequencing (NGS) has been utilized for high-volume, high-resolution genotyping of HLA loci at the DKMS Life Science Lab for over three years.
• This study focuses on experiences with genotyping HLA, CCR5, ABO, RHD, and KIR genes using direct amplicon sequencing on Illumina platforms.

Purpose of the Study:

• To report on the experiences and optimizations of amplicon-based NGS for high-volume HLA genotyping.
• To detail workflow improvements that enhance efficiency and robustness in tissue typing.

Main Methods:

• Direct amplicon sequencing on Illumina MiSeq and HiSeq 2500 instruments.
• Implementation of a multi-step workflow including nanofluidic chips and 4-primer amplicon tagging.
• Workflow optimizations such as split protocols for different DNA concentrations, use of phosphorothioate oligos, and statistical modeling for read yield prediction.

Main Results:

• Processed over 2.7 million samples from potential stem cell donors between 2013 and 2016.
• Achieved high-resolution typing for 98.9% of targeted HLA alleles (HLA-A, -B, -C, -DRB1, -DQB1, -DPB1).
• Reduced average per-locus repeat rates from 3.1% to 1.3% through workflow refinements.
• Discovered 1,919 novel HLA alleles from an analysis of 1.4 million donors.

Conclusions:

• Amplicon-based NGS HLA genotyping is a robust method for high-volume tissue typing of registry donors.
• Continuous optimization of workflow practices is crucial for improving the efficiency and reliability of short amplicon-based genotyping.
• The DKMS Life Science Lab's experience provides valuable insights into large-scale HLA genotyping using NGS.