Rapid, scalable and highly automated HLA genotyping using next-generation sequencing: a transition from research to diagnostics.

BACKGROUND: Human leukocyte antigen matching at allelic resolution is proven clinically significant in hematopoietic stem cell transplantation, lowering the risk of graft-versus-host disease and mortality. However, due to the ever growing HLA allele database, tissue typing laboratories face substantial challenges. In light of the complexity and the high degree of allelic diversity, it has become increasingly difficult to define the classical transplantation antigens at high-resolution by using well-tried methods. Thus, next-generation sequencing is entering into diagnostic laboratories at the perfect time and serving as a promising tool to overcome intrinsic HLA typing problems. Therefore, we have developed and validated a scalable automated HLA class I and class II typing approach suitable for diagnostic use. RESULTS: A validation panel of 173 clinical and proficiency testing samples was analysed, demonstrating 100% concordance to the reference method. From a total of 1,273 loci we were able to generate 1,241 (97.3%) initial successful typings. The mean ambiguity reduction for the analysed loci was 93.5%. Allele assignment including intronic sequences showed an improved resolution (99.2%) of non-expressed HLA alleles. CONCLUSION: We provide a powerful HLA typing protocol offering a short turnaround time of only two days, a fully integrated workflow and most importantly a high degree of typing reliability. The presented automated assay is flexible and can be scaled by specific primer compilations and the use of different 454 sequencing systems. The workflow was successfully validated according to the policies of the European Federation for Immunogenetics. Next-generation sequencing seems to become one of the new methods in the field of Histocompatibility.

New Aspects of the Virus Life Cycle and Clinical Utility of Next Generation Sequencing based HIV-1 Resistance Testing in the Genomic, the Proviral, and the Viral Reservoir of Peripheral Blood Mononuclear Cells.

BACKGROUND: Typically, genotypic resistance testing is recommended at the start of antiretroviral therapy and is even mandatory in cases of virologic failure. The material of choice is plasma viral RNA. However, in patients with low viremia (viral load < 500 copies/ml), resistance testing by population-based sequencing is very difficult. OBJECTIVE: Therefore, we aimed to investigate whether next generation sequencing (NGS) from proviral DNA and RNA could be an alternative. MATERIAL AND METHODS: EDTA blood samples (n = 36) from routine clinical viral load testing were used for the study. Viral loads ranged from 96 to 390,000 copies/mL, with 100% of samples having low viremia. Distribution of subtypes; A (n = 2), B (n = 16), C (n = 4), D (n = 2), G (1), CRF02 AG (n = 5), CRF01 AE (n = 5), undefined/mixed (n = 4). The extracted consensus sequences were uploaded to the Stanford HIV Drug Resistance Data Base and Geno2pheno for online analysis of drug resistance mutations and resistance factors. RESULTS: A total of 2476 variants or drug resistance mutations (DRMs) were detected with Sanger sequencing, compared with 2892 variants with NGS. An average of 822/1008 variants were identified in plasma viral RNA by Sanger or NGS sequencing, 834/956 in cellular viral RNA, and 820/928 in cellular viral DNA. CONCLUSION: Both methods are well suited for the detection of HIV substitutions or drug resistance mutations. Our results suggest that cellular RNA or cellular viral DNA is an informative alternative to plasma viral RNA for variant detection in patients with low viremia, as shown by the high correlation of variants in the different viral pools. We show that by using UDS, a plus of two DRMs per patient becomes visible, which can make a big difference in the assessment of the expected resistance behavior of the virus.