Abstract
Aim The HLA region is the most complex and polymorphic region in the genome making its genotyping in the clinic challenging. In recent years the continuous and partial characterization of new alleles has significantly increased the level of ambiguity using traditional genotyping techniques as a result requiring reflexive testing, sometimes extensive. Next Generation Sequencing (NGS) is an advanced method able to provide unambiguous results and eliminate the need for reflexive testing in the clinical laboratory. We aim to use NGS and the unique consensus generation algorithm of HLA Twin to resolve previously ambiguous or problematic samples. Methods The ability to capture the entire length of the HLA loci and analyze them by constructing whole gene consensus sequences using de novo assembly techniques makes it a very powerful tool for the identification of novel alleles, null alleles as well as the resolution of typings that traditional methods are not able to. Holotype HLA and HLA Twin were used to generate full-length consensus sequences for both alleles at every locus to fully characterize and unambiguously genotype a series of problematic and challenging samples. Results In this study, we present how HLA Twin’s analysis algorithms are able to accurately identify and resolve a variety of “tricky” results, such as the insertion of a 15-nucleotide sequence in an exon changing the structure of the resulting protein. In addition, de novo assembly is the only method that can detect alternative splice sites in intronic regions that may also change the resulting protein. Several features in HLA Twin such as the display of the amino acid sequence, the phasing track and the noise track (reads with systematic noise that have been filtered out during the analysis) are useful tools that facilitate the user’s confirmation of the findings. Conclusions NGS, and in particular Holotype HLA, is already being implemented for clinical typing successfully worldwide and provides increasing clarity into challenging samples.
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