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The development of next-generation sequencing (NGS) methods for HLA genotyping has already had an impact on the scope and precision of HLA research. In this study, allelic resolution HLA typing was obtained for 402 individuals from Cape Town, South Africa. The data were produced by high-throughput NGS sequencing as part of a study of T-cell responses to Mycobacterium tuberculosis in collaboration with the University of Cape Town and Stanford University. All samples were genotyped for 11 HLA loci, namely HLA-A, -B, -C, -DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRB3, -DRB4, and -DRB5. NGS HLA typing of samples from Cape Town inhabitants revealed a unique cohort, including unusual haplotypes, and 22 novel alleles not previously reported in the IPD-IMGT/HLA Database. Eight novel alleles were in Class I loci and 14 were in Class II. There were 62 different alleles of HLA-A, 72 of HLA-B, and 47 of HLA-C. Alleles A∗23:17, A∗43:01, A∗29:11, A∗68:27:01, A∗01:23, B∗14:01:01, B∗15:10:01, B∗39:10:01, B∗45:07, B∗82:02:01 and C∗08:04:01 were notably more frequent in Cape Town compared to other populations reported in the literature. Class II loci had 21 different alleles of DPA1, 46 of DPB1, 27 of DQA1, 26 of DQB1, 41 of DRB1, 5 of DRB3, 4 of DRB4 and 6 of DRB5. The Cape Town cohort exhibited high degrees of HLA diversity and relatively high heterozygosity at most loci. Genetic distances between Cape Town and five other sub-Saharan African populations were also calculated and compared to European Americans.

Background Genome-Wide Association Studies (GWAS) have produced strong evidence for association of Schizophrenia (SCZ) in the extended Major Histocompatibility Complex (chromosome 6q), including the Human Leukocyte Antigen (HLA) region. It has been reported that structural polymorphisms in the C4 gene can explain the signal in this region, but it has not been determined by direct high-resolution genotyping whether HLA alleles have independent effects. We have used a novel next-generation sequencing method to achieve HLA typing with 8-digit (4-field) resolution over classical HLA genes (A, B, C, DRB1, DRB3-5, DQA1, DQB1, DPA1 and DPB1) in cohorts of three ethnicities; and assayed C4 structural polymorphisms in a subset of the sample to permit imputation in the entire cohorts. These data permit analysis of the association of SCZ with HLA alleles while accounting for C4 variants. Methods Subjects included 21,592 individuals: SCZ cases/controls from the MGS and GPC cohorts (European-ancestry: 5,762 cases, 5,558 controls; African-American: 2,671 cases, 2,373 controls), and 1,670 Taiwan families (3,340 parents, 1,728 affected offspring). HLA genotyping was carried out using the Mia Fora NGS Flex HLA Typing Kit (Immucor), which uses long-range PCR to capture 11 HLA loci (the entire gene in most cases) followed by high-depth, multiplexed Illumina sequencing. C4 polymorphisms were assayed using ddPCR using published primers and calling algorithms, in 940 individuals representing all ancestries and genotyping platforms. Analyses are almost completed, including imputation of C4 structural alleles into all samples (HIBAG), followed by frequentist association tests of HLA alleles with and without conditioning on C4 alleles, taking ancestry covariates into account (PLINK). It is expected that imputation of HLA genotypes can also be improved for each ancestry based on the high-resolution genotyping of large cohorts. Results All analyses are in progress and will be completed prior to the Congress. Missing data rates across loci are ~1%, and Mendelian error rate in trios is low. C4 alleles can be imputed with high accuracy in all combinations of ancestry and genotyping platform. Imputation accuracy was over 90% for all HLA genes. Discussion The results of this study will provide important new information about whether HLA alleles have effects on SCZ risk, independent of their linkage disequilibrium with C4 structural polymorphisms. The study will also provide improved resolution of imputation of both C4 polymorphisms and of HLA alleles.

Dissecting the molecular basis of quantitative traits is a significant challenge and is essential for understanding complex diseases. Even in model organisms, precisely determining causative genes and their interactions has remained elusive, due in part to difficulty in narrowing intervals to single genes and in detecting epistasis or linked quantitative trait loci. These difficulties are exacerbated by limitations in experimental design, such as low numbers of analyzed individuals or of polymorphisms between parental genomes. We address these challenges by applying three independent high-throughput approaches for QTL mapping to map the genetic variants underlying 11 phenotypes in two genetically distant Saccharomyces cerevisiae strains, namely (1) individual analysis of >700 meiotic segregants, (2) bulk segregant analysis, and (3) reciprocal hemizygosity scanning, a new genome-wide method that we developed. We reveal differences in the performance of each approach and, by combining them, identify eight polymorphic genes that affect eight different phenotypes: colony shape, flocculation, growth on two nonfermentable carbon sources, and resistance to two drugs, salt, and high temperature. Our results demonstrate the power of individual segregant analysis to dissect QTL and address the underestimated contribution of interactions between variants. We also reveal confounding factors like mutations and aneuploidy in pooled approaches, providing valuable lessons for future designs of complex trait mapping studies.

Aim There is very limited information on the importance of DRB1 exon 1 variations in transplantation. Routinely, clinical laboratories do not test for exon 1 assuming it codes for the leader peptide which is cleaved off of the mature protein, and therefore not clinically important. Currently there are two CWD DRB1 alleles that differ in exon 1, DRB1∗12:01 and DRB1∗12:10, which can only be resolved by sequencing exon 1. We sought to interrogate exon 1 of DRB1 for the presence of the CWD allele, DRB1∗12:10, and to determine the HLA class II haplotypes associated with it in our local population. Methods The Immucor Mia Fora Flex 11 HLA Typing kit was used to genotype eight HSCT recipients and their unrelated donors, as well as seven additional patients. All 23 of these individuals were previously genotyped as DRB1∗12:01 using SSOP. Results All 23 individuals typed as DRB1∗12:01:01. No DRB1∗12:10 was identified in this limited cohort. Two haplotypes were identified for DRB1∗12:01:01: sixteen individuals (76%) were DRB1∗12:01:01, DRB3∗02:02:01, DQA1∗05:05:01, DQB1∗03:01:01, and five individuals (24%) were DRB1∗12:01:01, DRB3∗01:01:02, DQA1∗05:05:01, DQB1∗03:01:01. Two individuals could not have their DRB3∗01:01:01 and DRB3∗02:02:01 haplotypes identified. Conclusions The Immucor Mia Fora Flex 11 HLA Typing kit is unique among several commercial NGS kits in that it contains primers capable of sequencing exon 1 of DRB1. Traditionally, this region has been avoided because of the difficulty in primer design due to the very large 8000–10,000 bp intron 1. The CWD allele, DRB1∗12:10 originally identified in Korean population, differs from DRB1∗12:01 by one bp in exon 1, but since exon 1 is not routinely typed, the individual frequencies of these alleles are not known. Retrospective studies in Asian populations indicated that 12% of DRB1 alleles previously typed as 12:01 were in Fact 12:10. In our study, 100% of our cohort of 23 patients typed as DRB1∗12:01, which was found to be predominantly associated with DRB3∗02:02:01 (76%) and less frequently with DRB3∗01:01:02 (23%). This is in contrast to the reported preferential association of DRB1∗12:01 with DRB3∗01:01:02 (97%), while association with DRB3∗02:02:01 was very rare (2.1%). Retrospective studies may be helpful to determine the importance of exon 1 mismatches in HSCT.

The genomes of sulfate-reducing bacteria remain poorly characterized, largely due to a paucity of experimental data and genetic tools. To meet this challenge, we generated an archived library of 15,477 mapped transposon insertion mutants in the sulfate-reducing bacterium Desulfovibrio alaskensis G20. To demonstrate the utility of the individual mutants, we profiled gene expression in mutants of six regulatory genes and used these data, together with 1,313 high-confidence transcription start sites identified by tiling microarrays and transcriptome sequencing (5′ RNA-Seq), to update the regulons of Fur and Rex and to confirm the predicted regulons of LysX, PhnF, PerR, and Dde_3000, a histidine kinase. In addition to enabling single mutant investigations, the D. alaskensis G20 transposon mutants also contain DNA bar codes, which enables the pooling and analysis of mutant fitness for thousands of strains simultaneously. Using two pools of mutants that represent insertions in 2,369 unique protein-coding genes, we demonstrate that the hypothetical gene Dde_3007 is required for methionine biosynthesis. Using comparative genomics, we propose that Dde_3007 performs a missing step in methionine biosynthesis by transferring a sulfur group to O-phosphohomoserine to form homocysteine. Additionally, we show that the entire choline utilization cluster is important for fitness in choline sulfate medium, which confirms that a functional microcompartment is required for choline oxidation. Finally, we demonstrate that Dde_3291, a MerR-like transcription factor, is a choline-dependent activator of the choline utilization cluster. Taken together, our data set and genetic resources provide a foundation for systems-level investigation of a poorly studied group of bacteria of environmental and industrial importance., IMPORTANCE Sulfate-reducing bacteria contribute to global nutrient cycles and are a nuisance for the petroleum industry. Despite their environmental and industrial significance, the genomes of sulfate-reducing bacteria remain poorly characterized. Here, we describe a genetic approach to fill gaps in our knowledge of sulfate-reducing bacteria. We generated a large collection of archived, transposon mutants in Desulfovibrio alaskensis G20 and used the phenotypes of these mutant strains to infer the function of genes involved in gene regulation, methionine biosynthesis, and choline utilization. Our findings and mutant resources will enable systematic investigations into gene function, energy generation, stress response, and metabolism for this important group of bacteria.

Aim Africans represent the most genetically diverse population in the world, but have not been as well studied with respect to their HLA types compared to the inhabitants of western countries. Thus it was of interest to apply our high-throughput HLA sequencing technology to a cohort of 403 healthy adolescents from Cape Town, South Africa, as part of a study of T-cell responses to Mycobacterium tuberculosis in collaboration with Stanford University. Methods Cryopreserved PBMC samples from 403 adolescents were collected and shipped to Stanford University from Cape Town. High resolution HLA typing was performed using MIA FORA NGS kit and analysis software. The whole gene coverage included HLA-A, -B, -C, -DPA1, -DQA1, and -DQB1; all exons and introns for -DRB1 and -DRB3/4/5 except partial coverage for exon 6 and intron 1; and all exons and introns between exons 2 and 4 for HLA-DPB1. NGS sequencing libraries were prepared using a semi-automated protocol and sequenced in high-throughput format on the Illumina NextSeq platform. HLA allele candidates were computed and final HLA typing was confirmed using MIA FORA NGS analysis software. Results HLA typing revealed a unique population, including unusual haplotypes, and novel alleles not previously reported in the IMGT/HLA database. The frequency of HLA-A, -B, -DRB1 haplotypes in Cape Town were compared to those represented in the US National Marrow Donor Program. As might be expected, 14 of the top 16 haplotypes in the Cape Town cohort were observed in the African American cohort. However there were important exceptions, including a frequent haplotype found in African American but not observed in Cape Town and two frequent Cape Town haplotypes not observed in African American samples. Conclusion High resolution HLA typing is a powerful method to characterize HLA allele and haplotype diversity in population studies. C. Wang: Consultant; Company/Organization; Immucor. M. Li: Employee; Company/Organization; Immucor. M. Fukushima: Employee; Company/Organization; Immucor. R. Kuehn: Employee; Company/Organization; Immucor. S. Krishnakumar: Employee; Company/Organization; Immucor. M. Mindrinos: Employee; Company/Organization; Immucor.