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Assessing a single targeted next generation sequencing for human leukocyte antigen typing protocol for
Manish J Gandhi1, Deborah Ferriola2, Curt Lind2
1Mayo Clinic, Rochester MN, United States.
Human Immunology
|July 23, 2017
Summary
This study demonstrates a simplified next-generation sequencing (NGS) protocol for human leukocyte antigen (HLA) typing that is accurate and reproducible. The protocol is suitable for clinical testing by technologists with varied NGS experience.
Area of Science:
- Immunogenetics
- Molecular Biology
- Genomics
Background:
- Next-generation sequencing (NGS) offers a powerful tool for human leukocyte antigen (HLA) typing.
- A simplified NGS protocol was developed for the Illumina MiSeq platform to assess its accuracy and reproducibility.
Purpose of the Study:
- To evaluate the accuracy and reproducibility of a simplified NGS-based HLA typing protocol.
- To assess the performance of the protocol across different laboratories and technologists with varying NGS experience.
Main Methods:
- A standardized protocol involving long-range PCR, library preparation, and paired-end sequencing was used.
- Two indexing strategies (locus-specific and sample-specific) were compared.
- Sequence analysis was performed using Target HLA and NGSengine software.
Main Results:
- High average depth of coverage (1444 reads/locus) was achieved.
- Genotype accuracy exceeded 97% for pooled amplicons and 99% for individual amplicons.
- Inter-laboratory reproducibility was 99.7%, with minimal discordance attributed to cross-contamination.
Conclusions:
- The simplified NGS HLA typing protocol is accurate, reproducible, and scalable.
- The protocol is amenable to clinical laboratory settings and can be performed by technologists with diverse NGS experience.
