![]() The incorporation of unique molecular identifiers (UMIs) in the initial library preparation has become standard for single-cell analysis ( Islam et al., 2014 Chen et al., 2018). One approach to reducing raw sequencing errors with ONT sequencing is the attachment of random oligomers of DNA to DNA or RNA before PCR amplification. Short read platforms obtain reads with an accuracy approaching 99.99% however, ONT sequencers achieve between 88–94% accuracy, depending on the chemistry and flow cell used ( Metzker, 2010 Liu et al., 2021). Such an assay would enable improved virtual crossmatching and epitope analysis ( Garcia-Sanchez et al., 2020).Ī challenge with Oxford Nanopore Technologies (ONT) sequencers is the relatively higher error rate compared to traditional short-read sequencers. The main focus for rapid HLA typing has been the development of an assay that yields high-resolution HLA typing in the time necessary for deceased donor allocation. ![]() Long read technology is beneficial and applicable for HLA typing due to various advantages of the methodology, including shorter library preparation, real-time base calling, shorter sequencing times, and potential cost savings. The application of long-read technology to HLA typing has been successfully demonstrated by several laboratories using DNA or RNA ( Liu et al., 2018a Liu, 20212018a Lang et al., 2018 Montgomery et al., 2019 De Santis et al., 2020 Mosbruger et al., 2020). ![]() Short read platforms are characterized by sequencing reads between 50 and 300 bp, while long-read platforms can sequence reads from 150 to >2 Mb ( Metzker, 2010 Jain et al., 2018). Currently, there are two major NGS platforms: short- and long-read sequencing. The application of next-generation sequencing (NGS) to human leukocyte antigen (HLA) typing has greatly reduced the costs and time required to achieve high-resolution HLA typing ( Lind et al., 2010 Weimer et al., 2016 Cornaby et al., 2021). This study demonstrates a rapid high-resolution HLA typing assay using RNA-Seq that can provide accurate HLA genotyping and HLA allele-specific transcript expression in 7–8 h, a timeline short enough to perform the assay for deceased donors. In general, the expression of class II transcripts was lower than that of class I transcripts. Similar to previous studies, for the class I loci, patients demonstrated significantly lower expression of HLA-C than HLA-A and -B (Mann–Whitney U, p value = 0.0065 and p value = 0.0154, respectively). The assay demonstrated an excellent concordance rate of 99.7%. Our assay used NGSEngine to determine the HLA genotyping result and normalized mRNA transcript expression using Athlon2. Our study cohort was evaluated using a long-read RNA sequencing methodology to provide rapid HLA genotyping results and normalized HLA transcript expression. It is becoming increasingly clear that understanding the expression of patient HLA transcripts can provide additional benefits for many of these same patient groups. HLA typing provides essential results for stem cell and solid organ transplants, as well as providing diagnostic benefits for various rheumatology, gastroenterology, neurology, and infectious diseases.
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