![]() ![]() Sanger sequencing is being used to verify these novel positions. The consensus sequences on the 24 remaining alleles contained novel differences when compared to existing reference sequences in the IMGT/HLA Database. The consensus sequences also confirmed the presence and phasing of the variants within the nine novel alleles. The HLA types of 219 of these alleles immediately matched the types observed previously. 252 HLA class I alleles were expected from the 45 DNA samples when homozygous loci were considered. Results Sufficient numbers of reads were obtained for each locus of each sample to enable HLA allele calling (mean 145 range 47–385) with mean a QV > 75. Each of the forty-five DNA samples were amplified for HLA-A, -B and -C using generic primers tagged with a DNA barcode unique to that sample resulting in 135 barcoded amplicons. Whole gene amplicons were generated for the HLA class I genes. Nine of these DNA samples had previously been identified as having novel alleles at one of their HLA class I loci using standard HLA typing methods. Forty-five DNA samples were selected for HLA class I typing and were d derived from different DNA sources (blood, saliva and umbilical cord blood). Further experiments to determine multiplexing using DNA barcodes have shown that multiplexing up to 20 DNA samples for a single HLA locus or 8 DNA samples for multiple loci, is possible. Method Our previous experience of sequencing full-length HLA class I genes using SMRT® technology has shown that it has read length capabilities and accuracy amenable for definitive allele-level resolution HLA typing. The clinical impact of achieving this level of resolution HLA typing data is likely to considerable, particularly in applications such as organ and blood stem cell transplantation where matching donors and recipients for their HLA is of utmost importance.Īim To sequence entire HLA class I genes of 45 DNA samples in a single sequencing reaction using DNA-barcode-labelled primers and Single Molecule, Real-Time (SMRT®) DNA sequencing technology. This method has the potential to revolutionize the field of HLA typing. Eight novel genomic HLA class I sequences were identified, four were novel alleles, three were confirmed as genomic sequence extensions and one corrected an existing genomic reference sequence. The results showed that SMRT DNA sequencing technology was able to generate sequences that spanned entire HLA Class I genes that allowed for accurate allele calling. Seven DNA samples were typed for HLA-A, -B and -C. Here we report on the feasibility of using Pacific Biosciences' Single Molecule Real-Time (SMRT) DNA sequencing technology for high-resolution and high-throughput HLA typing. The use of higher resolution methods is often restricted due to cost and time limitations. Many high-throughput molecular HLA typing approaches are unable to determine the phase of observed DNA sequence polymorphisms, leading to ambiguous results. Allele-level resolution data at primary HLA typing is the ideal for most histocompatibility testing laboratories. ![]()
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