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Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons
Published on: August 29, 2014
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Very high resolution single pass HLA genotyping using amplicon sequencing on the 454 next generation DNA sequencers:
F Yamamoto1, B Höglund2, M Fernandez-Vina1
1Stanford University, Palo Alto, CA, USA.
Human Immunology
|June 4, 2015
Summary
Next-generation sequencing enhances high-resolution Human Leukocyte Antigen (HLA) genotyping. This improved assay offers greater accuracy and unambiguous results, potentially replacing current Sanger sequencing methods for clinical applications.
Area of Science:
- Immunogenetics
- Molecular Biology
- Genomics
Background:
- Next-generation sequencing (NGS) offers superior throughput, cost-effectiveness, and reduced ambiguity for Human Leukocyte Antigen (HLA) genotyping compared to Sanger sequencing.
- Existing HLA genotyping assays can face challenges with common alleles and null alleles, leading to genotype ambiguity.
Purpose of the Study:
- To describe an enhanced Roche 454 GS GType HLA genotyping assay for very high resolution (VHR) typing.
- To simplify the workflow for HLA genotyping and improve genotype assignment accuracy.
Main Methods:
- The enhanced assay incorporates 8 additional primer pairs to the original 14, enabling genotyping of 11 HLA loci.
- Workflow simplification strategies, including early amplicon pooling and the Fluidigm Access Array™, were investigated.
- Performance was evaluated using well-characterized cell lines and clinical samples, with results analyzed by Conexio Assign MPS software using genomic reference sequences.
Main Results:
- Testing on cell lines showed 98.4% concordance with 53% unambiguous calls.
- Testing on clinical samples demonstrated 98.7% concordance, with 58.2% unambiguous calls.
- The assay successfully resolved common alleles and excluded null alleles, reducing genotype ambiguity.
Conclusions:
- The amplicon-based VHR assay is a robust method for high-resolution HLA typing, suitable for replacing Sanger sequencing.
- Combined with software enhancements, this assay has the potential to achieve even higher resolution HLA typing for clinical applications.
