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HLA allele-level typing is mandatory for HCT patients and potential donors. Amplicon based Sanger sequencing is unable to detect HLA haplotypes with known gametic phase, resulting in various ambiguous combinations of several possible HLA genotypes. Resolution of these ambiguous combinations can only be done using, PCR-SSP or PCR-SSO, both of which are expensive and time consuming. NGS technology can process multiple samples, and allele assignment should be relatively simple and should decrease resolve ambiguous genotypes. Our laboratory routinely sequences approximately 100 samples per month for HLA-A, B, C, DRB1, DQB1 and DPB1. We validated the usefulness of NGS to resolve ambiguities and reduce the need for SSP and/or SSO. Commercially available KITs (Scisco Genetics) were used to analyze 110 samples with known HLA genotypes. We amplified exons 2, 3, and 4 of HLA Class I genes and exons 2 and 3 of HLA Class II genes. Phase between exons 2 and 3 for Class I were established by sequencing directly, while the sequences of the Class II exons were established by comparison with the IMGT HLA data base. Allele assignments were made using software developed by Scisco Genetics. We observed 100% concordance for HLA class II alleles, and 92% for Class I alleles. All discrepancies for Class I were due to failure to correctly assign one allele, but not the second allele (different from homozygotes). NGS was able to detect several recently identified HLA alleles, e.g. A ∗ 02:139 and C ∗ 04:119. Allele assignment was simplified and fast, but set up was the most laborious and time consuming. However, we are sure that liquid handling robots (Texas BioGene Inc) will be able to automate the pre-PCR and post-PCR steps. KIT can be used to detects HLA genes: such as HLA-DPA1, HLA-DQA1, or HLADRB3,4,5. We are in process of validating more samples, and changing our HLA class I protocol to achieve 100% concordance. We anticipate sharp declines in the need for PCR-SSP and PCR-SSO testing. The benefit of NGS for high resolution HLA typing should be significantly decreased costs, and shortened turnaround times. N. Wyatt: Employee; Company/Organization; Scisco Genetics. Stock Shareholder; Company/Organization; Scisco Genetics. D. Geraghty: Employee; Company/Organization; Scisco Genetics. Stock Shareholder; Company/Organization; Scisco Genetics. [ABSTRACT FROM AUTHOR]

Aim: Killer cell immunoglobulin-like receptor (KIR) complex is polygenic and polymorphic. Our PCR-SSP Multiplex detects the presence of 14 KIR genes in 4 PCR reactions, but genotype and Centromeric/telomeric (Cen/Tel) haplotype assignments are performed manually, which can be time-consuming when analyzing large data sets. The aim of this study was to validate a new real-time (RT)-PCR KIR genotyping Kit (LinkSeq KIR) using 40 samples. Methods: RT-PCR is a sensitive method, does not include post-PCR analysis steps and can be easily automated. LinkSeq KIR Kit with SYBR green uses a PCR-based method and melt curve analysis to determine KIR specificity. A separate and unique control well determined DNA quality. SureTyper software assigns KIR genotypes, as well as KIR Cen/Tel haplotypes. Results: KIR genotypes using the LinkSeq method agreed with previous typing results, except one 2DS2 gene and one 2DP1 gene were initially reported as negative. Upon review, software threshold settings were ignoring somewhat weaker positive signals. After adjusting software analysis settings, all samples were concordant. We were not able to detect several framework KIR genes for one sample: 2DL4, 3DP1 and 3DL1 and 3DS1 corresponding to genotype #194, ID from allelefrequencies.net which was observed in one person from South Africa. The LinkSeq method also confirmed this unusual genotype and haplotypes were assigned as: CenB/B, TelB/B. Conclusions: In summary, the Linkage RT-PCR KIR genotyping kit is specific and sensitive, the amount of DNA is comparable with our multiplexing method, and post-PCR analysis is completely automated. This is the main advantage of using RT-PCR for large studies. Suretyper also assigns haplotypes based on KIR location (Cooley et al 2010). Our recommendation was to adopt the RT-PCR test to detect gene copy-number, which is the key to assigning KIR haplotypes and distinguishing homozygotes from hemizygotes. [Copyright &y& Elsevier]