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Full-Length HLA Sequencing Improves Accuracy and Resolution for Community Resource

Monday, December 11, 2017

In a new publication, scientists from Anthony Nolan Research Institute and the UCL Cancer Institute present an in-depth analysis of the utility of SMRT Sequencing for Human Leukocyte Antigen (HLA) typing. They assessed more than 100 cell lines and found that PacBio long-read sequencing significantly improves the accuracy of HLA typing.

Single molecule real-time (SMRT®) DNA sequencing of HLA genes at ultra-high resolution from 126 International HLA and Immunogenetics Workshop cell lines” comes from lead author Thomas Turner, senior author Steven Marsh, and collaborators. The scientists implemented SMRT Sequencing to perform high-resolution HLA typing for 126 B-lymphoblastoid cell lines, including a group of 107 cell lines established in 1987 that is now an essential resource for the community. The goal of the present study was to increase the resolution of the reference sequences in the IMGT/ HLA database and improve standardization of HLA typing calls for these cell lines.

HLA genes — used to evaluate donor-recipient tissue match before organ transplant, as well as other immune-related traits — are among the most polymorphic in the genome. Characterizing them has been a challenge with short-read sequencing platforms, but recent efforts to perform full-length sequencing and phasing of the genes with PacBio long-read sequencing have generated impressive results. Indeed, the authors write, “Anthony Nolan’s Histocompatibility Laboratory now routinely uses SMRT sequencing for HLA typing.”

For this project, scientists carried out amplicon sequencing for full-length gene analysis of HLA class I genes and partial analysis of class II genes. In total, they sequenced 931 HLA alleles, with 96% yielding results that matched previously established HLA types for those cell lines. Of the few dozen discrepancies, Turner et al. discovered that 10 harbored novel alleles and 13 were different because of zygosity results, while many others included allele types not previously reported for those cell lines. Confirmation studies showed that these SMRT Sequencing results accurately resolved ambiguities and corrected errors in earlier HLA typing efforts. “We identified numerous discrepancies and novel intronic polymorphisms, extended several alleles to full genomic sequences, and confirmed the existence of some alleles identified by other researchers,” the team reports.

“The work presented here has further demonstrated the efficacy of SMRT sequencing to provide the highest resolution, unambiguous HLA typing data when full genes are sequenced,” the scientists conclude. “This knowledge ensures the continued usefulness of the reference cell line panel as a resource to the immunogenetics community in the age of next generation DNA sequencing.”

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