Detection and annotation of transposable element insertions and deletions on the human genome using nanopore sequencing.

Repetitive sequences represent about 45% of the human genome. Some are transposable elements (TEs) with the ability to change their position in the genome, creating genetic variability both as insertions or deletions, with potential pathogenic consequences. We used long-read nanopore sequencing to identify TE variants in the genomes of 24 patients with antithrombin deficiency. We identified 7 344 TE insertions and 3 056 TE deletions, 2 926 were not previously described in publicly available databases. The insertions affected 3 955 genes, with 6 insertions located in exons, 3 929 in introns, and 147 in promoters. Potential functional impact was evaluated with gene annotation and enrichment analysis, which suggested a strong relationship with neuron-related functions and autism. We conclude that this study encourages the generation of a complete map of TEs in the human genome, which will be useful for identifying new TEs involved in genetic disorders.

G protein-coupled receptor kinase 5 regulates thrombin signaling in platelets via PAR-1.

The interindividual variation in the functional response of platelets to activation by agonists is heritable. Genome-wide association studies (GWASs) of quantitative measures of platelet function have identified fewer than 20 distinctly associated variants, some with unknown mechanisms. Here, we report GWASs of pathway-specific functional responses to agonism by adenosine 5'-diphosphate, a glycoprotein VI-specific collagen mimetic, and thrombin receptor-agonist peptides, each specific to 1 of the G protein-coupled receptors PAR-1 and PAR-4, in subsets of 1562 individuals. We identified an association (P = 2.75 × 10-40) between a common intronic variant, rs10886430, in the G protein-coupled receptor kinase 5 gene (GRK5) and the sensitivity of platelets to activate through PAR-1. The variant resides in a megakaryocyte-specific enhancer that is bound by the transcription factors GATA1 and MEIS1. The minor allele (G) is associated with fewer GRK5 transcripts in platelets and the greater sensitivity of platelets to activate through PAR-1. We show that thrombin-mediated activation of human platelets causes binding of GRK5 to PAR-1 and that deletion of the mouse homolog Grk5 enhances thrombin-induced platelet activation sensitivity and increases platelet accumulation at the site of vascular injury. This corroborates evidence that the human G allele of rs10886430 is associated with a greater risk for cardiovascular disease. In summary, by combining the results of pathway-specific GWASs and expression quantitative trait locus studies in humans with the results from platelet function studies in Grk5-/- mice, we obtain evidence that GRK5 regulates the human platelet response to thrombin via the PAR-1 pathway.

Development and validation of a universal blood donor genotyping platform: a multinational prospective study.

Each year, blood transfusions save millions of lives. However, under current blood-matching practices, sensitization to non-self-antigens is an unavoidable adverse side effect of transfusion. We describe a universal donor typing platform that could be adopted by blood services worldwide to facilitate a universal extended blood-matching policy and reduce sensitization rates. This DNA-based test is capable of simultaneously typing most clinically relevant red blood cell (RBC), human platelet (HPA), and human leukocyte (HLA) antigens. Validation was performed, using samples from 7927 European, 27 South Asian, 21 East Asian, and 9 African blood donors enrolled in 2 national biobanks. We illustrated the usefulness of the platform by analyzing antibody data from patients sensitized with multiple RBC alloantibodies. Genotyping results demonstrated concordance of 99.91%, 99.97%, and 99.03% with RBC, HPA, and HLA clinically validated typing results in 89 371, 3016, and 9289 comparisons, respectively. Genotyping increased the total number of antigen typing results available from 110 980 to >1 200 000. Dense donor typing allowed identification of 2 to 6 times more compatible donors to serve 3146 patients with multiple RBC alloantibodies, providing at least 1 match for 176 individuals for whom previously no blood could be found among the same donors. This genotyping technology is already being used to type thousands of donors taking part in national genotyping studies. Extraction of dense antigen-typing data from these cohorts provides blood supply organizations with the opportunity to implement a policy of genomics-based precision matching of blood.

Publisher Correction: Whole-genome sequencing of a sporadic primary immunodeficiency cohort.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Whole-genome sequencing of a sporadic primary immunodeficiency cohort.

Primary immunodeficiency (PID) is characterized by recurrent and often life-threatening infections, autoimmunity and cancer, and it poses major diagnostic and therapeutic challenges. Although the most severe forms of PID are identified in early childhood, most patients present in adulthood, typically with no apparent family history and a variable clinical phenotype of widespread immune dysregulation: about 25% of patients have autoimmune disease, allergy is prevalent and up to 10% develop lymphoid malignancies1-3. Consequently, in sporadic (or non-familial) PID genetic diagnosis is difficult and the role of genetics is not well defined. Here we address these challenges by performing whole-genome sequencing in a large PID cohort of 1,318 participants. An analysis of the coding regions of the genome in 886 index cases of PID found that disease-causing mutations in known genes that are implicated in monogenic PID occurred in 10.3% of these patients, and a Bayesian approach (BeviMed4) identified multiple new candidate PID-associated genes, including IVNS1ABP. We also examined the noncoding genome, and found deletions in regulatory regions that contribute to disease causation. In addition, we used a genome-wide association study to identify loci that are associated with PID, and found evidence for the colocalization of-and interplay between-novel high-penetrance monogenic variants and common variants (at the PTPN2 and SOCS1 loci). This begins to explain the contribution of common variants to the variable penetrance and phenotypic complexity that are observed in PID. Thus, using a cohort-based whole-genome-sequencing approach in the diagnosis of PID can increase diagnostic yield and further our understanding of the key pathways that influence immune responsiveness in humans.

Multiple GYPB gene deletions associated with the U- phenotype in those of African ancestry.

BACKGROUND: The MNS blood group system is defined by three homologous genes: GYPA, GYPB, and GYPE. GYPB encodes for glycophorin B (GPB) carrying S/s and the "universal" antigen U. RBCs of approximately 1% of individuals of African ancestry are U- due to absence of GPB. The U- phenotype has long been attributed to a deletion encompassing GYPB exons 2 to 5 and GYPE exon 1 (GYPB*01N). STUDY DESIGN AND METHODS: Samples from two U-individuals underwent Illumina short read whole genome sequencing (WGS) and Nanopore long read WGS. In addition, two existing WGS datasets, MedSeq (n = 110) and 1000 Genomes (1000G, n = 2535), were analyzed for GYPB deletions. Deletions were confirmed by Sanger sequencing. Twenty known U- donor samples were tested by a PCR assay to determine the specific deletion alleles present in African Americans. RESULTS: Two large GYPB deletions in U- samples of African ancestry were identified: a 110 kb deletion extending left of GYPB (DEL_B_LEFT) and a 103 kb deletion extending right (DEL_B_RIGHT). DEL_B_LEFT and DEL_B_RIGHT were the most common GYPB deletions in the 1000 Genomes Project 669 African genomes (allele frequencies 0.04 and 0.02). Seven additional deletions involving GYPB were seen in African, Admixed American, and South Asian samples. No samples analyzed had GYPB*01N. CONCLUSIONS: The U- phenotype in those of African ancestry is primarily associated with two different complete deletions of GYPB (with intact GYPE). Seven additional less common GYPB deletion backgrounds were found. GYPB*01N, long assumed to be the allele commonly encoding U- phenotypes, appears to be rare.

Automated typing of red blood cell and platelet antigens: a whole-genome sequencing study.

BACKGROUND: There are more than 300 known red blood cell (RBC) antigens and 33 platelet antigens that differ between individuals. Sensitisation to antigens is a serious complication that can occur in prenatal medicine and after blood transfusion, particularly for patients who require multiple transfusions. Although pre-transfusion compatibility testing largely relies on serological methods, reagents are not available for many antigens. Methods based on single-nucleotide polymorphism (SNP) arrays have been used, but typing for ABO and Rh-the most important blood groups-cannot be done with SNP typing alone. We aimed to develop a novel method based on whole-genome sequencing to identify RBC and platelet antigens. METHODS: This whole-genome sequencing study is a subanalysis of data from patients in the whole-genome sequencing arm of the MedSeq Project randomised controlled trial (NCT01736566) with no measured patient outcomes. We created a database of molecular changes in RBC and platelet antigens and developed an automated antigen-typing algorithm based on whole-genome sequencing (bloodTyper). This algorithm was iteratively improved to address cis-trans haplotype ambiguities and homologous gene alignments. Whole-genome sequencing data from 110 MedSeq participants (30 × depth) were used to initially validate bloodTyper through comparison with conventional serology and SNP methods for typing of 38 RBC antigens in 12 blood-group systems and 22 human platelet antigens. bloodTyper was further validated with whole-genome sequencing data from 200 INTERVAL trial participants (15 × depth) with serological comparisons. FINDINGS: We iteratively improved bloodTyper by comparing its typing results with conventional serological and SNP typing in three rounds of testing. The initial whole-genome sequencing typing algorithm was 99·5% concordant across the first 20 MedSeq genomes. Addressing discordances led to development of an improved algorithm that was 99·8% concordant for the remaining 90 MedSeq genomes. Additional modifications led to the final algorithm, which was 99·2% concordant across 200 INTERVAL genomes (or 99·9% after adjustment for the lower depth of coverage). INTERPRETATION: By enabling more precise antigen-matching of patients with blood donors, antigen typing based on whole-genome sequencing provides a novel approach to improve transfusion outcomes with the potential to transform the practice of transfusion medicine. FUNDING: National Human Genome Research Institute, Doris Duke Charitable Foundation, National Health Service Blood and Transplant, National Institute for Health Research, and Wellcome Trust.

Validation of self-administered nasal swabs and postage for the isolation of Staphylococcus aureus.

Staphylococcus aureus carriers are at higher risk of S. aureus infection and are a reservoir for transmission to others. Detection of nasal S. aureus carriage is important for both targeted decolonization and epidemiological studies. Self-administered nasal swabbing has been reported previously, but the effects of posting swabs prior to culture on S. aureus yield have not been investigated. A longitudinal cohort study was performed in which healthy volunteers were recruited, trained in the swabbing procedure and asked to take weekly nasal swabs for 6 weeks (median: 3 weeks, range 1-6 weeks). Two swabs were taken at each sampling episode and randomly assigned for immediate processing on arrival to the laboratory (Swab A) or second class postage prior to processing (Swab B). S. aureus was isolated using standard methods. A total of 95 participants were recruited, who took 944 swabs (472 pairs) over a median of 5 weeks. Of these, 459 swabs were positive for S. aureus. We found no significant difference (P=0.25) between 472 pairs of nasal self-swabs processed immediately or following standard postage from 95 study participants (51.4 % vs. 48.6 %, respectively). We also provide further evidence that persistent carriers can be detected by two weekly swabs with high degrees of sensitivity [92.3 % (95 % CI 74.8-98.8 %)] and specificity [95.6 % (95 % CI 84.8-99.3 %)] compared with a gold standard of five weekly swabs. Self-swabbing and postage of nasal swabs prior to processing has no effect on yield of S. aureus, and could facilitate large community-based carriage studies.

Low-Density TaqMan® Array Cards for the Detection of Pathogens.

Real-time PCR assays have revolutionised diagnostic microbiology over the past 15 years or more. Adaptations and improvements over that time frame have led to the development of multiplex assays. However, limitations in terms of available fluorophores has meant the number of assays which can be combined has remained in single figures. This latter limitation has led to the focus tending to be on individual pathogens and their detection. This chapter describes the development of TaqMan® Array Cards (TACs), technology which allows the detection of multiple pathogens (up to 48 targets) from a single nucleic acid extract, utilising small volumes and real-time PCR. This in turn lends itself to a syndromic approach to infectious disease diagnosis. Using the examples of TACs we have developed in our own laboratory, as well as others, we explain the design, optimisation and use of TACs for respiratory, gastrointestinal and liver infections. Refinement of individual assays is discussed as well as the incorporation of appropriate internal and process controls onto the array cards. Finally, specific examples are given of instances where the assays have had a direct, positive impact on patient care.