High throughput HLA genotyping using 454 sequencing and the Fluidigm Access Array™ System for simplified amplicon...

Area of Science:

• Immunogenetics
• Genomics
• Molecular Biology

Background:

• Human leukocyte antigen (HLA) loci are highly polymorphic, making accurate genotyping challenging.
• Efficient HLA typing is crucial for unrelated bone marrow donor registries to ensure successful transplantation.
• Existing methods often require high sample throughput and low cost per sample.

Purpose of the Study:

• To develop and validate a high-resolution, high-throughput HLA genotyping method.
• To leverage next-generation sequencing and multiplex identifiers for accurate HLA allele identification.
• To assess the feasibility of this method for donor registry applications.

Main Methods:

• Utilized the 454 Genome Sequence (GS) FLX System with Conexio Assign ATF 454 software for sequencing.
• Employed the Fluidigm Access Array System for multiplexing 48 samples with unique multiplex identifiers (MIDs).
• Generated 2304 parallel PCRs using a 4-primer system incorporating MIDs and 454 adaptor sequences for amplicon generation.

Main Results:

• Achieved 100% concordance with known genotypes for 192 samples using 8 primer pairs and 96 MIDs in a single GS FLX run.
• Obtained an average of 166 sequence reads per amplicon for initial genotyping.
• Successfully performed high-resolution genotyping on 96 samples using 14 primer pairs, yielding an average of 173 reads per amplicon.

Conclusions:

• The described method provides high-resolution, high-throughput HLA genotyping.
• This approach is accurate and efficient for typing multiple HLA loci.
• The technology is suitable for applications in bone marrow donor registries, enhancing donor selection processes.