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Next generation DNA sequencing is used to determine the HLA-A, -B, -C, -DRB1, -DRB3/4/5, and -DQB1 assignments of 1009 unrelated volunteers for the unrelated donor registry in The Netherlands. The analysis characterizes all HLA exons and introns for class I alleles; at least exons 2 to 3 for HLA-DRB1; and exons 2 to 6 for HLA-DQB1. Of the distinct alleles present, there are 229 class I and 71 class II; 36 of these alleles are novel. The majority (approximately 98%) of the cumulative allele frequency at each locus is contributed by alleles that appear three or more times. Alleles encoding protein variation outside of the antigen recognition domains are 0.6% of the class I assignments and 5.3% of the class II assignments.

Coronavirus disease 2019 (COVID-19), driven by SARS-CoV-2, is a severe infectious disease that has become a global health threat. Vaccines are among the most effective public health tools for combating COVID-19. Immune status is critical for evaluating the safety and response to the vaccine, however, the evolution of the immune response during immunization remains poorly understood. Single-cell RNA sequencing (scRNA-seq) represents a powerful tool for dissecting multicellular behavior and discovering therapeutic antibodies. Herein, by performing scRNA/V(D)J-seq on peripheral blood mononuclear cells from four COVID-19 vaccine trial participants longitudinally during immunization, we revealed enhanced cellular immunity with concerted and cell type-specific IFN responses as well as boosted humoral immunity with SARS-CoV-2-specific antibodies. Based on the CDR3 sequence and germline enrichment, we were able to identify several potential binding antibodies. We synthesized, expressed and tested 21 clones from the identified lineages. Among them, one monoclonal antibody (P3V6-1) exhibited relatively high affinity with the extracellular domain of Spike protein, which might be a promising therapeutic reagent for COVID-19. Overall, our findings provide insights for assessing vaccine through the novel scRNA/V(D)J-seq approach, which might facilitate the development of more potent, durable and safe prophylactic vaccines.

Next generation DNA sequencing is used to determine the HLA-A, -B, -C, -DRB1, and -DQB1 assignments of 1472 unrelated volunteers for the unrelated donor registry in Argentina. The analysis characterized all HLA exons and introns for class I alleles; at least exons 2, 3 for HLA-DRB1; and exons 2 to 6 for HLA-DQB1. Of the distinct alleles present, there are 330 class I and 98 class II. The majority (~98%) of the cumulative allele frequency at each locus is contributed by alleles that appear at a frequency of at least 1 in 1000. Fourteen (18.2%) of the 77 novel class I and II alleles carry nonsynonymous variation within their exons; 52 (75.4%) class I novel alleles carry only single, apparently random, nucleotide variation within their introns/untranslated regions. Alleles encoding protein variation not usually detected by typing focused only on the exons encoding the antigen recognition domain are 1.0% of the class I assignments and 7.3% of the class II assignments (predominantly DQB1*02:02:01, DQB1*03:19:01, and DRB1*14:54:01). Updates to the common and well documented list of alleles include 10 alleles previously thought to be uncommon but that are found at least 30 times. Five locus haplotypes estimated using the expectation-maximization algorithm as present 3 or more times total 187. While the known HLA diversity continues to increase, the conservation of known allele sequences is remarkable. Overall, the HLA diversity observed in the Argentinian population reflects its European and Native American ancestry.

The 2014 Ebola outbreak in West Africa highlighted the worldwide public health threat posed by this virus and the urgent need for an Ebola vaccine. A novel recombinant adenovirus type 5 vector-based Ebola vaccine (Ad5-EBOV), based on the 2014 Zaire Guinea epidemic strain, was developed in China. A good safety profile and robust immune response elicited by Ad5-EBOV were confirmed in phase 1 and phase 2 clinical trials. Nonetheless, clinical studies of this Ebola vaccine are still at an early stage and there are still no solid efficacy data for humans. For efficacy evaluation and quality control of Ad5-EBOV, the cellular and humoral immune responses in BALB/c mice vaccinated with Ad5-EBOV were examined at various time points. ELISpot and flow cytometric analysis showed that EBOV glycoprotein (GP)-specific T cell responses were detectable early in the first week after infection and by week 4 had increased to maximum levels, which lasted through week 6. During week 1, high titers of EBOV GP-specific antibodies were found (geometric mean [GM], 1783). These titers peaked at week 10 (GM, 26,214) and lasted to 6 months (GM, 1,351). The titer of neutralizing antibodies based on pseudovirus assays also increased over time to peak at 1:16 in one mouse and 1:8 in nine mice during week 6, before decreasing to zero by week 12. These results suggest that BALB/c mice can be used to evaluate the effectiveness of Ad5-EBOV, and that the cellular immune response and humoral immune response can be used as indicators to evaluate vaccine effectiveness. Rapid determination of such methods and indicators is critical for the evaluation of Ebola vaccine efficacy, and can provide effective quality control for Ad5-EBOV.

Objective: Mutations of the cyclin F (CCNF) gene were recently identified to be associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in Western and Japanese populations. The rare protein-altering variants frequency is 0.6 to 3.3% in ALS and FTD from these diverse geographic populations while no systematic analysis of CCNF variants were conducted in the Chinese population.Methods: We screened all exons of CCNF in a cohort of 269 cases (including 181 ALS and 88 FTD) from Mainland China using Sanger sequencing.Results: A rare heterozygous variant (c.481G > A, p.G161R) was detected in a sporadic ALS case with a frequency of 0.6%, while no mutation was identified in patients with FTD. The same variant was also found in a sporadic ALS patient from America.Conclusions: Our result indicates that the mutation of CCNF is rare in patients with ALS and FTD from Mainland China.

Aim 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. Methods 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. Results 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. Conclusions 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.

HLA class I assignments were obtained at single genotype, G-level resolution from 98 855 volunteers for an unrelated donor registry in the United States. In spite of the diverse ancestry of the volunteers, over 99% of the assignments at each locus are common. Within this population, 52 novel alleles differing in exons 2 and 3 are identified and characterized. Previously reported alleles with incomplete sequences in the IPD-IMGT/HLA database (n = 519) were selected for full gene sequencing and, from this sampling, another 27 novel alleles are described.

Recently, the coiled‑coil‑helix‑coiled‑coil‑helix domain 2 (CHCHD2) gene was identified as a possible causative gene for Parkinson's disease (PD). Three other neurodegenerative diseases, Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), share significant overlaps with PD in clinical phenotypes, pathological features and genetic heredities, and it is still unclear whether CHCHD2 variants could explain these three diseases. The present study screened all exons of the CHCHD2 gene in a total of 780 patients (511 AD, 181 ALS and 88 FTD) and 500 healthy controls from the Chinese Han population. Two missense variants, 5C>T (Pro2Leu) and 238A>G (Ile80Val), were identified in five unrelated patients with AD while no mutations were observed in patients with ALS or FTD. These mutations have been reported as low‑frequency variants in the ExAC database with frequencies of 0.0075 and 0.000025. Pro2 Leu, however, was also detected in controls and was confirmed to have no significant association with the risk for AD; Ile80Val was not detected in any normal controls, suggesting that the CHCHD2 gene may be associated with AD in the Chinese Han population.

Two novel HLA class II alleles, DRB4*03:01N and DQB1*03:276N, containing large deletions were identified during routine typing. Extraction of DNA encompassing the deletions was carried out with a panel of capture oligonucleotides followed by whole genome amplification. Next generation DNA sequencing was then used to characterize the sequences. DRB4*03:01N has a 16 kilobase pair deletion stretching upstream from intron 2 toward centromeric DRB8. DQB1*03:276N has two deletions separated by 844 nucleotides. The first deletion (3.7 kilobase pairs) is upstream of intron 1 and the second deletion removes 3.3 kilobase pairs further upstream towards centromeric DQA2.

The filamentous fungus Aspergillus oryzae 3.042 (Chinese strain) is a close relative of A. oryzae RIB40 (Japanese strain), which is the important agent used for soy sauce fermentation. The genome of A. oryzae 3.042 was sequenced and compared with A. oryzae RIB40 in an attempt to understand why different soy sauce flavors are produced by these strains. The A. oryzae 3.042 chromosome is 36,547,279 bp and contains 11,399 protein-encoding genes. MUMmer analysis revealed that the genomes of A. oryzae 3.042 and RIB40 are mostly collinear. Genome sequence data and comparative analysis of the two strains identified several strain-specific genes that encode putative proteins involved in cell growth, salt tolerance, environmental resistance and flavor formation. A. oryzae 3.042 showed stronger potential for mycelial growth. Some genes unique to A. oryzae RIB40 were related to salt tolerance, especially genes for K + transport, while others were associated with ester formation and amino acid metabolism, which likely contribute to flavor formation. In conclusion, comparative genome analysis provided insights into the different genetic traits of the two A. oryzae strains. The unique genes that we found in A. oryzae would make sense to the soy sauce fermentation.

HLA-A, -B, -C, -DRB1, -DQB1 assignments were obtained for 374 pairs of African American mothers and their umbilical cord blood units (CBU) by DNA sequencing. An algorithm developed by the National Marrow Donor Program was used to assign 1122 haplotypes by segregation. 70% of the haplotypes carried assignments at all five loci. In the remainder, alleles at various loci, most often DQB1 in 48% of the haplotypes with a missing assignment, could not be assigned due to sharing of both alleles by mother and CBU. There were 652 haplotypes carrying a unique combination of alleles at the five loci; the majority (74%) were singletons. Novel B~C and DRB1~DQB1 associations were observed. The results demonstrate the genetic diversity in this population and provide validation for a publically available tool for pedigree analysis. Our observations underscore the need for procurement of increased numbers of units in the national cord blood inventory in order to identify matching donors for all patients requiring hematopoietic stem cell transplantation.

Traditional DNA-based typing focuses primarily on interrogating the exons of human leukocyte antigen (HLA) genes that form the antigen recognition domain (ARD). The relevance of mismatching donor and recipient for HLA variation outside the ARD on hematopoietic stem cell transplantation (HSCT) outcomes is unknown. This study was designed to evaluate the frequency of variation outside the ARD in 10 of 10 (HLA-A, -B, -C, -DRB1, -DQB1) matched unrelated donor transplant pairs (n = 360). Next-generation DNA sequencing was used to characterize both HLA exons and introns for HLA-A, -B, -C alleles; exons 2, 3 and the intervening intron for HLA-DRB1 and exons only for HLA-DQA1 and -DQB1. Over 97% of alleles at each locus were matched for their nucleotide sequence outside of the ARD exons. Of the 4320 allele comparisons overall, only 17 allele pairs were mismatched for non-ARD exons, 41 for noncoding regions and 9 for ARD exons. The observed variation between donor and recipient usually involved a single nucleotide difference (88% of mismatches); 88% of the non-ARD exon variants impacted the amino acid sequence. The impact of amino acid sequence variation caused by substitutions in exons outside ARD regions in D-R pairs will be difficult to assess in HSCT outcome studies because these mismatches do not occur very frequently.

African trypanosomes cause human sleeping sickness and livestock trypanosomiasis in sub-Saharan Africa. We present the sequence and analysis of the 11 megabase-sized chromosomes of Trypanosoma brucei . The 26-megabase genome contains 9068 predicted genes, including ∼900 pseudogenes and ∼1700 T. brucei –specific genes. Large subtelomeric arrays contain an archive of 806 variant surface glycoprotein (VSG) genes used by the parasite to evade the mammalian immune system. Most VSG genes are pseudogenes, which may be used to generate expressed mosaic genes by ectopic recombination. Comparisons of the cytoskeleton and endocytic trafficking systems with those of humans and other eukaryotic organisms reveal major differences. A comparison of metabolic pathways encoded by the genomes of T. brucei, T. cruzi , and Leishmania major reveals the least overall metabolic capability in T. brucei and the greatest in L. major . Horizontal transfer of genes of bacterial origin has contributed to some of the metabolic differences in these parasites, and a number of novel potential drug targets have been identified.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons of the brain, brainstem and spinal cord. To date, mutations in more than 30 genes have been linked to the pathogenesis of ALS. Among them, SOD1, FUS and TARDBP are ranked as the three most common genes associated with ALS. However, no mutation analysis has been reported in central-southern China. In this study, we sequenced SOD1, FUS and TARDBP in a central-southern Chinese cohort of 173 patients with ALS (15 familial ALS and 158 sporadic ALS) to detect mutations. As a result, five missense mutations in SOD1, namely, p.D101N, p.D101G, p.C111Y, p.N86S and p.V87A, were identified in three unrelated familial probands and three sporadic cases; two mutations in FUS were found in two unrelated familial probands, including an insertion mutation (p.P525_Y526insY) and a missense mutation (p.R521H); no variants of TARDBP were observed in patients. Therefore, SOD1 mutations were present in 20.0% of familial ALS patients and 1.9% of sporadic ALS patients, while FUS mutations were responsible for 13.3% of familial ALS cases and TARDBP mutations were rare in either familial or sporadic ALS cases. This study broadens the known mutational spectrum in patients with ALS and further demonstrates the necessity for genetic screening in ALS patients from central-southern China.

The genome of Candida versatilis was sequenced to understand its characteristics in soy sauce fermentation. The genome size of C. versatilis was 9.7 Mb, the content of G + C was 39.74 %, scaffolds of N50 were 1,229,640 bp in length, containing 4711 gene. There were predicted 269 tRNA genes and 2201 proteins with clear function. Moreover, the genome information of C. versatilis was compared with another salt-tolerant yeast Zygosaccharomyces rouxii and the model organism Saccharomyces cerevisiae. C. versatilis and Z. rouxii genome size was close and both smaller than 12.1 for the Mb of S. cerevisiae. Using the OrthoMCL protein, three genomes were divided into 4663 groups. There were about 3326 homologous proteins in C. versatilis, Z. rouxii and S. cerevisiae.

In developing countries, trauma patients and neonates are vulnerable to Staphylococcus aureus (S. aureus) and Clostridium tetani infections. It has been suggested that a combined vaccine against the two infections may be a reliable and cost‑effective strategy. Previous studies have indicated that the S. aureus surface protein A (SasA) and the C fragment of tetanus neurotoxin (TeNT‑Hc) may be suitable candidates for a vaccine against S. aureus and tetanus infections, respectively. In the present study, mice were immunized with a combined vaccine containing SasA and TeNT‑Hc, which induced a robust immune response to both antigens, and mutual interference between SasA and TeNT‑Hc was not observed. In the S.aureus challenge model, the combined vaccine fully protected BALB/c mice against lethal intraperitoneal challenges with 3x109 colony‑forming units of a methicillin‑resistant S. aureus USA300 strain. In the TeNT challenge model, the combined vaccine conferred complete protection against a lethal dose of (2x103) xLD50 tetanus toxin. These results implied that SasA and TeNT‑Hc promising components for a combined vaccine against S. aureus and tetanus infections.

Anthrax toxin causes anthrax pathogenesis and expression levels of ANTXR2 (anthrax toxin receptor 2) are strongly correlated with anthrax toxin susceptibility. Previous studies found that ANTXR2 transcript abundance varies considerably in individuals of different ethnic/geographical groups, but no eQTLs (expression quantitative trait loci) have been identified. By using 3C (chromatin conformation capture), CRISPR-mediated genomic deletion and dual-luciferase reporter assay, gene loci containing cis-regulatory elements of ANTXR2 were localized. Two SNPs (single nucleotide polymorphism) at the conserved CREB-binding motif, rs13140055 and rs80314910 in the promoter region of the gene, modulating ANTXR2 promoter activity were identified. Combining these two regulatory SNPs with a previously reported SNP, rs12647691, for the first time, a statistically significant correlation between human genetic variations and anthrax toxin sensitivity was observed. These findings further our understanding of human variability in ANTXR2 expression and anthrax toxin susceptibility.

The frequencies of alleles of killer cell immunoglobulin-like receptor genes, KIR3DL3 and KIR3DL2, and the carrier frequency of KIR2DL4 alleles have been determined from a population of African Americans (n = 100) by DNA sequencing of the coding regions. Fifty alleles of KIR3DL3 were observed with the most frequent, KIR3DL3*00901 (13%). KIR3DL2 was also diverse; 32 alleles with KIR3DL2*00103 the most frequent (17%). For KIR2DL4, of the 18 alleles observed, one allele, KIR2DL4*00103, was found in 64 of the 100 individuals. Thirty-six novel alleles encoding a total of 28 unique receptors are described. Pairwise comparisons among all of the alleles at each locus suggest a predominance of synonymous substitutions. The variation at all three framework loci fits a neutral model of evolution.

Aim The aim of this study was to complete full gene sequences for previously reported HLA class I sequences for the IPD-IMGT/HLA database. Methods We identified 569 class I alleles in ∼ 99,000 individuals typed for a U.S. donor registry that had incomplete sequences in the IPD-IMGTHLA database. The alleles had been typed by a combined Sanger SBT-SSOP workflow focused on exons 2 + 3. Single locus alleles were amplified with primers annealing in the 5’ and 3’ UTR and the sequences of exons and introns determined with an Illumina Miseq using an in-house protocol. Results Most (423, 74%) of the 569 alleles were found in a single cell. The majority of the class I full length sequences were identical to the most similar HLA allele (e.g., C*01:70 differed by only a single exon 2 nucleotide when compared to C*01:02:01:01). Two novel HLA-A alleles with exon 2/3 variation were not detected by SBT/SSO due to inability to phase key polymorphic nucleotides [A*03:150, *26:01:01G vs. A*03:01:01:01 + A*26:149 (new); A*02:01:55 + *33:03:01G vs. A*02:01:01:01 + *33:03:33 (new)]. Overall, new alleles were found in 18 (3.2%) samples; most in the targeted incomplete allele. Most (14, 78%) showed variation in intron and UTR regions. Some of the incomplete alleles (49/569) could not be characterized because the allele combination did not allow phasing of the sequences (e.g., C*07:01:01:01 + *07:27:01 had no polymorphic nucleotides within a span of 2298 bp (introns 2–7)). Conclusions Class I gene sequences outside exons 2 + 3 are conserved. Genotypes with novel alleles can be missed by typing assays that do not provide comprehensive phasing. Increasing the knowledge of full sequences for all alleles will improve the accuracy of HLA typing. Download : Download high-res image (153KB) Download : Download full-size image J.S. Ng: 4. Scientific/Medical Advisor; Company/Organization; Georgetown University. C.K. Hurley: 4. Scientific/Medical Advisor; Company/Organization; Georgetown University.

Aim Long range next generation sequencing was used to characterize the HLA class I (HLA-A,B,C) and class II (DRB1, DRB3/4/5, DQB1) alleles of a hematopoietic stem cell registry population (N = 643) from The Netherlands (Matchis). Methods The in-house typing protocol included several new strategies to reduce errors, add automation, and balance the yield of all loci when 96 samples are sequenced in one 600 cycle paired-end Illumina MiSeq sequence run. Results Of the 22 new class I alleles, three contain a nonsynonymous substitution in an exon (1, 4 or 7). The remainder are single nucleotide intron variants. Thirty-two class I alleles are incomplete in the IMGT/HLA database. The majority of the alleles in the population are common; the three most common alleles at each locus are listed in the table. Alleles not previously listed as common or well-documented include mainly 4th field variations; exceptions included single copies of B*51:20, C*07:100, and DRB1*04:92. Alleles encoding nonsynonymous substitutions compared to the primary allele in the G group include, as examples, A*23:17 (1 vs. 20 A*23:01:01:01), B*44:27:01 (1 vs 83 B*44:02:01:01), C*07:06 (3 vs 190 C*07:01:01:01), DRB1*14:54:01 (44 vs 3 DRB1*14:01:01), DQB1*02:02:01 (84 vs 175 DQB1*02:01:01). Non-expressed alleles were not observed. DRB3/4/5 associations were identified. Conclusions Long-range sequencing of HLA alleles from world-wide populations will continue to build our understanding of HLA diversity and its impact on the immune response. Download : Download high-res image (231KB) Download : Download full-size image C.K. Hurley: 7. Other (Identify); Company/Organization; Intellectual property sequencing Thermo Fisher.

The KIR2DS4 variants differ in exon 5 and play a role in hematopoietic stem cells transplantation (HSCT). A sequence-based testing (SBT) and TOPO TA cloning system identifying and distinguishing alleles of the KIR2DS4 gene was established and applied to a total of 150 Chinese-Han individuals: 75 patients received T-cell-depleted HSCT and their unrelated donors. The majority (139) of the 150 samples (92.7%) were positive for KIR2DS4. Four of the nine known KIR2DS4 alleles, KIR2DS4 *00101, *003,*004, and *007, were identified. In the haplotype A/A group, a higher risk of acute graft-versus-host disease (aGVHD) was seen when the donor carried two full-length KIR2DS4 alleles (RR 9.0 [95% CI 1.2-66.9], P = 0.010). Our findings suggested that the expression of full-length 2DS4 (*001) in A/A group may contribute to a worse clinical outcome after URD-HSCT. These data would be beneficial for the selection of suitable donors.

KIR2DL5 alleles were physically linked to alleles at adjacent KIR loci to define this region of KIR haplotypes in 55 gene-positive random African Americans. The majority carried KIR2DL5B. Three KIR2DL5A and six KIR2DL5B alleles that have been previously described and 11 novel KIR2DL5 alleles were identified by DNA sequencing. Novel alleles included variation that may impact promoter activity; two alleles carried nonsynonymous coding region variation. Based on linkage with KIR2DS1, KIR2DS3, KIR2DS5, KIR2DL2, KIR2DL3, and KIR3DS1 alleles, seven haplotypes of KIR2DL5A and 23 haplotypes of KIR2DL5B were observed. The phylogenetic relationships among the KIR2DL5 alleles predicted their association with either KIR2DS3 (six alleles) or KIR2DS5 (seven alleles). All of the KIR2DL5A alleles were linked either to KIR3DS1*01301 or KIR3DS1*049N. The majority of the KIR2DL5B alleles were linked to seven KIR2DL2 alleles; two were linked to a novel allele of KIR2DL3. These findings underscore the diversity of KIR haplotypes present in this population.

The five two-domain stimulatory KIR genes carried by 100 random African Americans were characterized by DNA sequencing of genomic DNA covering the majority of coding exons. The frequency of individual loci was similar to that found in European Americans, with the exception of a reduced frequency for KIR2DS1 in African Americans. New alleles were identified at the KIR2DS1 (*008), KIR2DS2 (*006), KIR2DS3 (*00104, *00105, *00106, *004), KIR2DS4 (*00103, *00104, *009, *011, *012, *013), and KIR2DS5 (*006, *007, *00801, *00802, *009) loci. The distribution of alleles at each locus was similar to that found in a European American population except for KIR2DS5. KIR2DS5 exhibits a single allele in European Americans; the same allele is found at a reduced frequency (41% of gene-positive individuals), accompanied by KIR2DS5*006 (18%), KIR2DS5*007 (26%), and six other alleles (25%), in African Americans.

Several overlapping amplicons were used to obtain the sequence of genomic DNA covering most of the coding regions of KIR3DL1 and KIR3DS1 from a family and 77 bone marrow transplant patients and their unrelated donors. Alleles 3DL1*00101 and *002 were most frequently observed in addition to 12 other known 3DL1 alleles. A single 3DS1 allele, 3DS1*01301, was identified in the 31 of 32 individuals carrying this gene. Two new alleles, 3DL1*01702 and 3DS1*058, were characterized. Three samples appeared to carry the duplicated killer cell immunoglobulin-like receptor (KIR) haplotype observed in other studies based on the presence of 3DS1 and two 3DL1 alleles. Additionally, one sample appeared to carry a novel KIR haplotype containing one 3DL1 and two 3DS1 alleles.

Genomic sequencing was used to characterize most of the coding regions of the five two-domain stimulatory killer cell immunoglobulin-like receptor (KIR) loci from 80 unrelated, primarily Caucasian, individuals. Specific loci were present in from 26% (KIR2DS3) to 98% (KIR2DS4) of individuals. The number of known alleles present varied from one (KIR2DS1, KIR2DS5) to five (KIR2DS4). The frequencies of loci and alleles were similar to observations made in populations of European and Asian ethnicities. New alleles were found at 2DS1 (*00202, *00302, *005, *006, *007) and 2DS4 (*008) loci.

Several copies of genes belonging to three multigene families present in the genome of Trypanosoma cruzi were sequenced and comparatively analyzed across six different strains of the parasite belonging to the T. cruzi I lineage (Colombiana, Silvio X10 and Dm28c), the T. cruzi II lineage (Esmeraldo and JG) and a hybrid strain (CL Brener). For all three gene families analyzed, our results support the division in T. cruzi I and II lineages. Furthermore, in agreement with its hybrid nature, sequences derived from the CL Brener clone clustered together with T. cruzi II sequences as well as with a third group of sequences. Paralogous sequences encoding Amastin, an amastigote surface glycoprotein and TcAG48, an antigenic RNA binding protein, which are clustered in the parasite genome, present higher intragenomic variability in T. cruzi II and CL Brener strains, when compared to T. cruzi I strains. Paralogous sequences derived from the TcADC gene family, which encode various isoforms of adenylyl cyclases and are dispersed throughout the T. cruzi genome, exhibit similar degree of variability in all strains, except in the CL Brener strain, in which the sequences were more divergent. Several factors including mutation rates and gene conversion mechanisms, acting differently within the T. cruzi population, may contribute to create such distinct levels of sequence diversity in multigene families that are clustered in the T. cruzi genome.

Exons 2-9 of KIR3DL3 alleles were characterized by genomic DNA sequencing in two families and in 78 bone marrow transplant samples. Several strategies were used to isolate single alleles for characterization and to resolve alternative allele combinations. We describe 17 novel 3DL3 alleles carried by 30 individuals. Compared with the most closely matched alleles, the new alleles differ by from one to three nucleotides and from zero to three amino acids. The majority of the substitutions were shared with other 3DL3 alleles although three novel polymorphic codons, 151 (exon 4), 327 (exon 7) and 352 (exon 9), are described. Of the 36 different 3DL3 alleles detected in the transplant population, the three most common alleles accounting for 47% of the total were KIR3DL3*00101 (13.5%), KIR3DL3*003 (21.2%) and KIR3DL3*00901 (12.2%).

Using paired-end sequences from bacterial artificial chromosomes, we have constructed high-resolution synteny and rearrangement breakpoint maps among human, mouse, and rat genomes. Among the >300 syntenic blocks identified are segments of over 40 Mb without any detected interspecies rearrangements, as well as regions with frequently broken synteny and extensive rearrangements. As closely related species, mouse and rat share the majority of the breakpoints and often have the same types of rearrangements when compared with the human genome. However, the breakpoints not shared between them indicate that mouse rearrangements are more often interchromosomal, whereas intrachromosomal rearrangements are more prominent in rat. Centromeres may have played a significant role in reorganizing a number of chromosomes in all three species. The comparison of the three species indicates that genome rearrangements follow a path that accommodates a delicate balance between maintaining a basic structure underlying all mammalian species and permitting variations that are necessary for speciation.

Aim The goal of this study was to update the 2012 common and well documented (CWD) allele list using data from 3940 class I and 4124 class II assignments obtained by next generation sequencing (NGS) of two registry (USA and Argentina) populations and 360 donor-recipient pairs from the CIBMTR repository. Methods DNA was prepared from blood, buccal swabs, or cell lines. HLA-A, -B, -C alleles were amplified with primers annealing in the 5’ and 3’ UTRs. The sequences of exons and introns were determined by next generation sequencing (NGS) with an Illumina Miseq using an in-house protocol and Conexio Assign software analysis. Class II NGS sequencing focused on exons 2 and 3; however, some alleles had additional exons and introns characterized. Alleles that were on the well-documented list and alleles not considered CWD based on Mack et al. Tissue Antigens 2013 were identified. Results The majority of alleles detected had previously been designated as common. For example, from the Argentine registry samples, 92% of 2944 HLA-A alleles identified were common. In the total study population, 77 class I alleles previously designated as WD and 97 alleles not defined as C or WD were observed from 1 to >5 times. Thirty-seven of the 174 class I alleles occurred greater than 5 times, e.g., previously undefined A*02:17:02, B*44:02:01:03, and C*17:01:01:05. The majority (90%) of the 19 not CWD alleles were intron variants e.g., A*03:01:01:05, B*39:01:01:03, C*06:02:01:03. Forty-five class II alleles fell into the WD or not defined categories; 29% occurred >5 times. Class II alleles occurring >5 times included DRB1*04:06:02, DQB1*03:01:01:03 and DPB1*04:02:01:02. Conclusions The NGS data expand our knowledge of the frequency of specific full length HLA alleles. The majority of the uncommon alleles characterized in this study are synonymous variations of common alleles.

Aim The aim of this study was to use next generation DNA sequencing (NGS) to characterize full length HLA class I gene diversity in 1472 volunteers for the unrelated donor registry in Argentina. Methods DNA was prepared from blood. HLA-A, -B, -C alleles were amplified with primers annealing in the 5′ and 3′ UTRs. The sequences of exons and introns were determined by NGS with an Illumina Miseq using an in-house protocol and Conexio Assign software analysis. Analysis used the IPD-IMGT/HLA database from October 2016. Results Volunteers were recruited from 20 of the 23 provinces of Argentina and from its federal capital. The majority (80%) came from the central region of the country. Of the 2944 alleles identified at each locus, the majority have been designated as common (e.g., 92% HLA-A alleles) (Mack et al. Tissue Antigens 2013) and approximately 1% were not found in the IPD-IMGT/HLA database. A total of 79 different allele sequences were identified at the HLA-A locus with similar numbers at HLA-B and -C. The majority of the HLA proteins can be distinguished by sequencing only the antigen recognition domain (ARD)-encoding exons. For example, 94% of the HLA-A alleles could have been detected solely based on the sequence of the ARD-encoding exons. The remaining alleles differed only outside of these exons by either silent variation occurring mainly in introns (6%) or by nonsynonymous variation ( Conclusions NGS has allowed us to evaluate the frequency and characteristics of class I DNA sequence variation outside of the ARD-encoding exons. The additional variation detected is predominantly in noncoding regions. While NGS offers a strategy to achieve single genotype resolution, the amount of additional information gained related to HLA matching for transplantation is minimal.

Aim The aim of this study was to characterize the complete class I gene sequences of new class I alleles found in a population study of 1472 volunteers from the Argentina unrelated donor registry. Methods HLA-A, -B, -C alleles were amplified with primers annealing in the 5’ and 3’ UTRs and the sequences of exons and introns determined by next generation sequencing (NGS) with an Illumina Miseq using an in–house protocol and Conexio Assign software analysis. Initially class I and II amplicons were combined for sequencing; single locus sequencing of a second PCR amplicon was used to confirm variation. Allele sequences were submitted to the IPD-IMGT/HLA database. Results 1% of the 2944 alleles identified at each class I locus were novel. Three novel alleles were found multiple times. The majority (92%) differed by only a single nucleotide substitution from the known allele. Variants in noncoding regions predominated (79%) (T1). None of the intron variants appeared to impact RNA splicing motifs. The distribution of novel substitutions among introns is listed in T2. Unique substitutions, not shared by any other known allele at the locus, were found in 71% HLA-A, 58% HLA-B and 67% HLA-C novel alleles. Conclusions Sixty-six novel class I alleles were identified. The majority of substitutions were found in introns and appear to be random rather than resulting from gene conversion events. Download : Download high-res image (295KB) Download : Download full-size image Download : Download high-res image (73KB) Download : Download full-size image

Aim This study used next generation sequencing (NGS) in 1472 healthy volunteers from an Argentinian registry to characterize class II gene diversity. Complications of using NGS for these regions arise from large introns, different length alleles, regions with few polymorphisms, and incomplete sequences in the IPD-IMGT/HLA database. Methods Amplicons were generated to include exons 2–6 of HLA-DQB1, and exons 2–3 of -DRB1 and -DPB1 using DNA extracted from blood. Samples were combined and sequenced on an Illumina MiSeq with an in-house library construction protocol. Conexio Assign was used to identify alleles based on the IPD-IMGT/HLA database from October 2016. Full length gene sequencing of select samples was performed with two overlapping amplicons for HLA-DPB1 and -DRB1. Results The majority of the alleles identified at each locus were common with ⩽1% of alleles per gene designated as well-documented or with frequency not defined. With only exons 2–3 sequenced, 6% of HLA-DRB1 assignments and 11% of -DPB1 were ambiguous. When additional exons were sequenced, most showed the common allele; however, DPB1∗104:01 was seen in 16% of samples typed as DPB1∗03:FNVX. For HLA-DRB1, preferential amplification due to differences in allele size and presence of pseudogenes led to certain alleles with low read depth and poor sequence quality, in particular DRB1∗04. Analysis of HLA-DQB1 introns proved difficult due to insertions and deletions that poorly aligned to the reference. Intron 2 of HLA-DPB1 is large and has few variations between certain combinations of alleles that were not phased in 14% of samples. These were not resolved with full length sequencing. Conclusions Although conditions still need to be optimized, NGS of HLA class II is a powerful tool in determining genetic diversity. HLA-DRB1 ambiguities would be reduced by 82% with the initial sequencing of exons 2–4; however, longer amplicons reduce read depth. Addition of primers to maximize amplification of low amplifying alleles may be necessary to increase read depth. Alternate methodology such as sequencing of single DNA molecules is necessary to solve the ambiguities for alleles that are not phased. These proposed changes will lead to more successful sequencing of HLA class II. Even so, these complications make NGS challenging when applied to high volume typing.

Aspergillus oryzae is a common fungus for traditional fermentation in Asia, such as spirit, soybean paste, and soy sauce fermentation. We report the 36.7-Mbp draft genome sequence of A. oryzae 100-8 and compared it to the published genome sequence of A. oryzae 3.042.

An effective approach, using the plasmid YCplac33-GHK, is developed to construct a ploidy series of Saccharomyces cerevisiae. YCplac33-GHK harbors the HO gene under the control of galactose-inducible promoter and KanMX4 as the selective marker. The simple method can solve the problem of industrial applications of strains with resistance genes.

The immune responses of natural killer cells are regulated, in part, by killer cell immunoglobulin-like receptors (KIR). The 16 closely-related genes in the KIR gene system have been diversified by gene duplication and unequal crossing over, thereby generating haplotypes with variation in gene copy number. Allelic variation also contributes to diversity within the complex. In this study, we estimated allele-level haplotype frequencies and pairwise linkage disequilibrium statistics for 14 KIR loci. The typing utilized multiple methodologies by four laboratories to provide at least 2x coverage for each allele. The computational methods generated maximum-likelihood estimates of allele-level haplotypes. Our results indicate the most extensive allele diversity was observed for the KIR framework genes and for the genes localized to the telomeric region of the KIR A haplotype. Particular alleles of the stimulatory loci appear to be nearly fixed on specific, common haplotypes while many of the less frequent alleles of the inhibitory loci appeared on multiple haplotypes, some with common haplotype structures. Haplotype structures cA01 and/or tA01 predominate in this cohort, as has been observed in most populations worldwide. Linkage disequilibrium is high within the centromeric and telomeric haplotype regions but not between them and is particularly strong between centromeric gene pairs KIR2DL5~KIR2DS3S5 and KIR2DS3S5~KIR2DL1, and telomeric KIR3DL1~KIR2DS4. Although 93% of the individuals have unique pairs of full-length allelic haplotypes, large genomic blocks sharing specific sets of alleles are seen in the most frequent haplotypes. These high-resolution, high-quality haplotypes extend our basic knowledge of the KIR gene system and may be used to support clinical studies beyond single gene analysis. © 2012 Vierra-Green et al.

Aim Traditional DNA-based typing focuses primarily on interrogating the exons of HLA genes that form the ARD (peptide-binding region that interacts with T cell and killer immunoglobulin-like receptors). The relevance of mismatching outside the ARD on hematopoietic stem cell transplantation (HCT) outcomes is unknown. This study was designed to evaluate the frequency of variation outside the ARD in well matched transplant pairs. Methods 360 HLA-A, B, C, DRB1, and DQB1 high resolution matched donor (D)-recipient (R) pairs were selected based on availability of retrospective Sanger-based sequencing and DNA from the CIBMTR Repository. The majority of the population was self-identified Caucasian (80%). Next generation DNA sequencing was performed on the Illumina MiSeq platform and interpreted with Connexio Assign MPS. Class I gene sequences covered 5 ′ UTR-3 ′ UTR; DRB1, intron 1-intron 3; DQA1 5 ′ UTR-exon 4; DQB1, intron 1-3 ′ UTR. DQ noncoding regions were not evaluated. Results The majority (98.1%) of the HLA-A, B, C, DRB1 D-R allele pairs are matched for sequences outside the ARD exons: 0.5% differ in non-ARD exons, 1.9% differ in noncoding regions. A small number (0.2%) differ within ARD exons. Mismatches in non-ARD exons varies from 0.7% for HLA-C and DQA1 to 0% DQB1; noncoding variation ranges from 2.8% for HLA-C to 1.3%, HLA-B and DRB1. Within non-ARD exons, both nonsynonymous (16 allele pairs) and silent (2) variation is present. Intron variation is minor, usually a single nucleotide. Conclusions This is the first study to evaluate the genetic variation and characterize mismatching outside of the ARD in a cohort of HLA-matched donor-recipient pairs. The paucity of exonic mismatches outside of the ARD is striking. Intronic variation is more common but would not contribute to an alloreactive mismatch. At present, it does not appear to be necessary to increase the resolution of HLA typing beyond the ARD in selecting a matched donor except in cases of common non-expressed variants within G-group assignments. The impact of amino acid sequence variation caused by substitutions in exons outside ARD regions in D–R pairs will be difficult to assess in HCT outcome studies since it does not occur very frequently. Further study is warranted to confirm these findings in larger and more diverse cohorts.

DNA sequencing is a powerful technique for identifying allelic variation within the natural killer (NK) cell immunoglobulin-like receptor genes. Because of the relatively large size of the KIR genes, each locus is amplified in two or more overlapping segments. Sanger sequencing of each gene from a preparation containing one or two alleles yields a sequence that is used to identify the alleles by comparison with a reference database.

Sir, Data recently published in Brain indicate that the CHCHD10 gene (NM_213720.2) plays an important role in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) (Bannwarth et al. , 2014; Johnson et al. , 2014; Muller et al. , 2014; Dols-Icardo et al. , 2015; Marroquin et al. , 2015). The CHCHD10 gene is located on chromosome 22q.11.23 and encodes a protein enriched at cristae junctions in mitochondria. Mutations in this gene cause mitochondrial dysfunction, which leads to disease. The study is the first report proposing that mitochondrial dysfunction contributes to the pathogenesis of ALS and FTD. The prevalence of the CHCHD10 mutation has been reported in a number of nationalities, including German (2.3%), French (2.6%), Spanish (0.68%) and Italian (1%) populations (Chaussenot et al. , 2014; Muller et al. , 2014; Chio et al. , 2015; Dols-Icardo et al. , 2015). However, no mutation analysis has been performed in Asian populations, and most variants have been on the ALS or ALS-FTD spectrum. Here, we present data showing that the CHCHD10 mutation is common in patients with the pure FTD phenotype in China. Patients with ALS ( n = 165) and FTD ( n = 65) were recruited at the outpatient clinic of Xiangya Hospital, China. The patients with ALS did not have mutations in SOD1 , FUS , C9orf72 and TARDBP . Additionally, the patients with FTD did not have any variants of MAPT , GRN and C9orf72 . We screened all of the exons in the CHCHD10 gene using Sanger sequencing. The analyses also included 500 unaffected individuals with matched geographical ancestry as healthy controls. This study was approved by the ethics committee of Xiangya Hospital, Central South University in China. …

Natural killer cell immunoglobulin-like receptor (KIR) haplotype-specific DNA fragments were sequenced to identify centromeric and telomeric allele level haplotype structures and their frequencies from 76 unrelated individuals with European ancestry. Analysis was simplified by redefining the 5’ boundary of the centromeric KIR gene cluster to include only exons 7-9 of KIR3DL3. Three consensus allele level haplotypes were identified for a centromeric gene presence/absence structure designated as Cen-A1. KIR3DL3*00201 (ex 7-9)—KIR2DL3*001—KIR2DL1*00302 was the most frequent (37.5%) centromeric structure. Single consensus haplotypes were observed for haplotype structures Cen-B1 and Cen-B2. Six Tel-A1 and two Tel-B1 consensus haplotypes were observed; the most prevalent (23.0%) was KIR2DL4*00102—KIR3DL1*002—KIR2DS4*00101—KIR3DL2*002. A small number of nucleotide substitutions (≤3) in the coding regions of the functional KIR genes created microvariants of the consensus haplotypes. Eight less common haplotype structures were also detected. Four carried hybrid genes formed during gene deletion events, two carried an insertion with a 2DL5/3DP1 fusion gene, and two included a very large insertion. These data show that the KIR gene complex is composed of a limited number of conserved allele level centromeric and telomeric haplotypes that have diversified by mutation, recombination within a locus, and unequal crossing over.

To understand the osmo-adaptation mechanism in Torulopsis versatilis (T), we investigated the salt-tolerant gene HOG1 from the wild-type and a salt-tolerant mutant strain (T5) constructed using genome shuffling. The HOG1 genes from T and T5 were sequenced and revealed several mutations had occurred. The expression level of T5HOG1 was stronger than that of THOG1, indicating a reason for the increase of salt-tolerance in T. versatilis. Moreover, overexpression of T5HOG1 and THOG1 improved the tolerance of salt in Saccharomyces cerevisiae. Identification and overexpression of THOG1 and T5HOG1 from the wild-type T. versatilis and the mutant T. versatilis, respectively, play an important role for the osmo-adaption mechanism of the T. versatilis used in soy-sauce fermentation.

KIR3DL1 and KIR3DS1 allele frequencies were determined by DNA sequencing of the complete coding regions from 100 random unrelated African Americans. Alleles 3DL1*01501 (29 individuals) and 3DL1*01502 (23 individuals) were most frequently observed in addition to 21 other known alleles and 7 new alleles: KIR3DL1*01503, *03102, *064, *065, *066, *067, and *068. Two of the new alleles (KIR3DL1*064, *065) created additional variants of the chimeric KIR3DL1/KIR3DL2 gene. KIR3DS1*01301 (94% of the gene positive individuals) and KIR3DS1*049N (11%) were identified in the 18 individuals carrying this gene. Three individuals appeared to carry a killer cell immunoglobulin-like receptor haplotype with KIR3DL1/S1 duplication. The profile of alleles resembles that found in African populations but also shows signs of admixture.

KIR2DL1 alleles were identified by DNA sequencing of the coding region from amplified genomic DNA from 100 random African Americans. The majority of individuals (97%) carried a KIR2DL1 locus. Allele KIR2DL1*00302 was found in 68% of individuals but KIR2DL1*00401, *002, *00303, *006, and *007 were also frequent. Eleven new alleles were described: KIR2DL1*00403, *01101, *01102, *012, *013N, *014, *015, *016, *017, *018, and *019. Nine of the novel alleles encoded amino acid substitutions located throughout the receptor; one allele carried a stop codon in the exon encoding the first extracellular domain.

Killer cell immunoglobulinlike receptors (KIRs) are a diverse family of receptors on natural killer cells. The KIR2DS4-deleted variant differs from the normal KIR2DS4 sequence by a 22-bp deletion in exon 5, which causes a frame shift, yielding a truncated KIR2DS4 protein with loss of the transmembrane and cytoplasmic domains of the full-length KIR2DS4 protein. A sequence-based testing and TOPO TA cloning system identifying and distinguishing alleles of the KIR2DS4 gene was established. The method was applied to 150 Chinese Han individuals: 75 patients who received T-depleted hematopoietic stem cells transplantation and their unrelated donors, establishing frequencies of 2DS4 allele and KIR gene within the local population. A majority (139) of the 150 samples (92.7%) were positive for KIR2DS4. The ratio of deleted to nondeleted versions of KIR2DS4 was approximately 1:2. Three KIR2DS4 novel alleles were identified. Forty-four percent individuals carried two group A haplotypes. The 17 KIR gene loci were found in 21.3%-100% in the population. Our findings suggest that the Chinese Han population is distinct in KIR gene frequencies and 2DS4 allele frequencies in comparison with some other populations.

PA-binding domain of LF (LFn) or PA-binding domain of EF (EFn) is the anthrax protective antigen (PA) binding domain of anthrax lethal factor (LF) or edema factor (EF). Here we show the development of a novel anthrax toxin inhibitor, fusion protein of N-terminal 27 amino acids deletion of LFn (Δ27LFn) and EFn. In a cell model of intoxication, fusion protein of Δ27LFn and EFn (Δ27LFn–EFn) was a 62-fold more potent toxin inhibitor than LFn or EFn, and this increased activity corresponded to a 39-fold higher PA-binding affinity by Biacore analysis. More importantly, Δ27LFn–EFn could protect the highly susceptible Fischer 344 rats from anthrax lethal toxin challenge. This work suggested that Δ27LFn–EFn has the potential as a candidate therapeutic agent against anthrax. Structured summary MINT- 7014735 , MINT- 7014747 , MINT- 7014761 : PA63 (uniprotkb: P13423 ) and LF (uniprotkb: P15917 ) bind (MI: 0407 ) by surface plasmon resonance (MI: 0107 )

Genome shuffling can improve complex phenotypes; however, there are several obstacles towards its broader applicability due to increased complexity of eukaryotic cells. Here, we describe novel, efficient and reliable methods for genome shuffling to increase ethanol production of Saccharomyces cerevisiae. Using yeast sexual and asexual reproduction by itself, mutant diploid cells were shuffled through highly efficient sporulation and adequate cross among the haploid cells, followed by selection on the special plates. The selected strain obtained after three round genome shuffling not only distinctly improved the resistance to ethanol, but also, increased ethanol yield by up to 13% compared with the control.

Sequencing of polymerase chain reaction (PCR)-amplified genomic DNA encompassing the putative proximal promoter and the coding region was used to identify KIR2DL5 alleles from 77 unrelated Caucasian individuals. PCR and sequencing were used to link each new allele to its neighboring KIR locus to identify 2DL5A or 2DL5B loci. Allele 2DL5A*001 was found in 24 of the 37 2DL5 positive individuals; 2DL5B*0020101 and 2DL5A*0050101 were also observed. Two new alleles, 2DL5B*008 and 2DL5B*009, contained substitutions altering the amino acid sequence of the leader and transmembrane region, respectively. Two other novel alleles, 2DL5B*0020102 and 2DL5A*0050102, contained alterations of the 5′ upstream region, bringing the number of unique promoter sequences to six. Promoter activity of the alleles was compared using luciferase reporter assays. Our results support those recently published, in which the promoter of 2DL5B*0020101 was shown to be more active in vitro compared to 2DL5A*001, and also provide additional information about the transcriptional activity of the promoters of the newly characterized alleles related to two altered transcription factor binding sites.

Sequencing of PCR amplified genomic DNA including most of the coding region was used to identify killer cell immunoglobulin-like receptor 2DL1 alleles from three families and 77 bone marrow transplant patients and donors. Alleles 2DL1*00302 and *002 were frequently observed in addition to two other known alleles and four new alleles, 2DL1*00402, 2DL1*007, 2DL1*008, and 2DL1*009.

Aim We explored the impact of using next generation sequencing (NGS) of the entire gene in 300 samples carrying potential new or rare HLA class I alleles. Methods Samples had been previously typed for exons 2 + 3 by SBT sequence based typing (Sanger) and/or by (SSO) sequence specific oligoprobes. Targeted class I loci were amplified using locus-specific PCR primers described by Hozomichi et al. Sonication was used to obtain 400 base pair fragments; an Illumina True Seq Nano kit was used for end repair and adaptor/index ligation. Libraries were pooled and paired end sequences were obtained with a MiSeq. AssignTM MPS software (Connexio, Genomics) was used for analysis. Results Of the 600 alleles sequenced, 69 (11.5%) novel alleles (HLA-A: 27, B: 31, C: 11) differed in the antigen recognition domain (ARD)-encoding exons 2 and/or 3. Ten additional alleles (1.7 %) carried previously unobserved differences in non-ARD-encoding exons (exon 1:2, exon 4:5, exon 5:2, exon 7:1). Thirty three alleles (5.5%) appeared to exhibit novel sequences only in introns. At HLA-A∗03:01:01:01 a novel insertion of 19 bp was observed in intron 2 in two different cells. One hundred and fifty seven (26%) alleles had incomplete sequences in the IMGT/HLA data base, of which most of these (121) had intron sequences identical to the closest related allele. The remaining 331 alleles were primarily common and well documented alleles. Conclusion NGS provided complete, unambiguous, high resolution allele assignments. In this more comprehensive analysis, there was less variation from the IMGT reference database than expected. Most of the introns of alleles with incomplete sequences were conserved when compared to their putative ancestral allele. L. Hou: Other (Identify); Company/Organization; Intellectual property. B. Tu: Other (Identify); Company/Organization; Intellectual property. C. Masaberg: Other (Identify); Company/Organization; Intellectual property. B. Beduhn: Employee; Company/Organization; National Marrow Donor Program. D. Goodridge: Employee; Company/Organization; Connexio Genomics. J. Ng: Other (Identify); Company/Organization; Intellectual property. C.K. Hurley: Other (Identify); Company/Organization; Intellectual property.

Aim We have developed an allele-level resolution next generation sequencing (NGS) strategy for HLA class I alleles and used it to explore HLA diversity in a registry donor population. Methods Long-range amplification of full-length HLA-A, -B, -C loci was performed in separate polymerase chain reactions using DNA from a buccal swab. Amplicons from one individual were then pooled and sheared by sonication. A library was constructed using Illumina’s TruSeq Nano kit and the DNA fragments tagged with one unique index combination. Libraries from 96 individuals were combined and sequenced simultaneously in a single 500 cycle (V2) paired-end run using an Illumina MiSeq. Data analysis used Conexio Genomics AssignTM MPS software. Results The typing protocol was validated with 552 donor registry samples previously typed by Sanger sequencing and probe hybridization at single genotype G level resolution, yielding a concordance of 99%. NGS yielded primarily single allele-level genotypes. Excluding amplification failures and inability to obtain an NGS assignment (4% loci), 32 discrepant NGS allele assignments were attributed to poor PCR amplification or loss of reads during assembly by software. Complete exon sequences identified 53 known A alleles (48 common or well-documented (CWD)), 89 B alleles (83 CWD), 52 C alleles (47 CWD). Ten potential new alleles with exon variation outside of exons 2 + 3 and over 25% of samples with potential intron variation require further study. The ability to make assignments based on specific regions of the sequence (e.g., exons only) was critical so that typings could be rapidly transmitted to the registry prior to a more comprehensive analysis of the diversity. Conclusions The protocol for NGS of class I alleles provides accurate results and should facilitate high volume high resolution typing of donor registry populations. The extent of new variation in exons is limited but the potential for intron variation is high. C. Hurley: Other (Identify); Company/Organization; Intellectual property.

Based on unusual probe hybridization patterns, two human leukocyte antigen (HLA)-A, five HLA-B, and two HLA-C alleles (A*240208, A*3211Q; B*1822, B*3938Q, B*4606, B*4607N, B*5139; Cw*0321, Cw*0734) were identified in individuals from the Singapore Bone Marrow Donor Program and Singapore Cord Blood Bank. Eight of the nine alleles encode amino acid substitutions altering the antigen-binding region including three alleles with changes altering a cysteine at codon 164 (A*3211Q, B*3938Q, B*4607N). This substitution either eliminates a key disulfide bond or results in a stop codon, both likely affecting the expression of the HLA molecules. Only one allele (A*240208) carries a synonymous substitution.