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• W4234408059 abstract "Sanger-based DNA sequencing of exons 2 + 3 of HLA class I alleles from a heterozygote frequently results in two or more alternative genotypes. In order to reduce the time and effort required to produce a single high resolution HLA genotype, samples were typed in parallel by Sanger sequencing and probe hybridization to obtain G level typing resolution. The panel of probes used was designed to yield high to intermediate resolution typing and to provide the phasing necessary to resolve common alternative genotypes. Class I assignments from more than 30,000 samples from registry donors were typed using this strategy supported by an in-house data merger and management software. To monitor accuracy and resolution, 552 samples were retyped by a next generation full length gene sequencing (NGS) strategy. More than 95% of the class I assignments were at the level of a single G-level genotype compared to ∼26% for European Americans when probe data were not included. The work load was reduced by at least 70% by eliminating secondary sequencing assays and by dramatically reducing the time needed for data review since the combined method addresses the weaknesses of each single method. Typing accuracy was increased. 99% of Sanger + probe typing results were concordant with NGS; all 32 allele discrepancies were attributed to weaknesses in the NGS data. Furthermore, NGS also demonstrated that only 0.9% of the G level assignments resulted in different full length protein sequences (e.g., C∗07:01:01G yielded C∗07:18 with NGS). The combined method had a rapid turnaround time compared with NGS and previous methods applied alone. Our combined method routinely provides biologically relevant typing resolution at the level of the antigen recognition domain. It can be applied to both single sample or to large volume typing supporting either bone marrow or solid organ transplantation using technology currently available in many HLA laboratories and is cost effective when compared with current NGS kits. B. Tu: Other (Identify); Company/Organization; Intellectual property. C. Masaberg: Other (Identify); Company/Organization; Intellectual property. J. Lee: Employee; Company/Organization; Thermo Fisher.D. Behm:Employee; Company/Organization; Data Blueprint.J. Sells:Employee; Company/Organization; Data Blueprint.P. Tausch: Employee; Company/Organization; Thermo Fisher.L. Hou:Other (Identify); Company/Organization; Intellectual property.J. Ng:Other (Identify); Company/Organization; Intellectual property.C.K. Hurley:Other (Identify); Company/Organization; Intellectual property." @default.
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• W4234408059 date "2015-10-01" @default.
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• W4234408059 title "Combining one step Sanger sequencing with phasing probe hybridization for class I typing yields rapid, g-group resolution defining 99% of unique full length protein sequences" @default.
• W4234408059 doi "https://doi.org/10.1016/j.humimm.2015.07.080" @default.
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