A quality control portal for sequencing data deposited at the European genome-phenome archive.

Since its launch in 2008, the European Genome-Phenome Archive (EGA) has been leading the archiving and distribution of human identifiable genomic data. In this regard, one of the community concerns is the potential usability of the stored data, as of now, data submitters are not mandated to perform any quality control (QC) before uploading their data and associated metadata information. Here, we present a new File QC Portal developed at EGA, along with QC reports performed and created for 1 694 442 files [Fastq, sequence alignment map (SAM)/binary alignment map (BAM)/CRAM and variant call format (VCF)] submitted at EGA. QC reports allow anonymous EGA users to view summary-level information regarding the files within a specific dataset, such as quality of reads, alignment quality, number and type of variants and other features. Researchers benefit from being able to assess the quality of data prior to the data access decision and thereby, increasing the reusability of data (https://ega-archive.org/blog/data-upcycling-powered-by-ega/).

The European Genome-phenome Archive in 2021.

The European Genome-phenome Archive (EGA - https://ega-archive.org/) is a resource for long term secure archiving of all types of potentially identifiable genetic, phenotypic, and clinical data resulting from biomedical research projects. Its mission is to foster hosted data reuse, enable reproducibility, and accelerate biomedical and translational research in line with the FAIR principles. Launched in 2008, the EGA has grown quickly, currently archiving over 4,500 studies from nearly one thousand institutions. The EGA operates a distributed data access model in which requests are made to the data controller, not to the EGA, therefore, the submitter keeps control on who has access to the data and under which conditions. Given the size and value of data hosted, the EGA is constantly improving its value chain, that is, how the EGA can contribute to enhancing the value of human health data by facilitating its submission, discovery, access, and distribution, as well as leading the design and implementation of standards and methods necessary to deliver the value chain. The EGA has become a key GA4GH Driver Project, leading multiple development efforts and implementing new standards and tools, and has been appointed as an ELIXIR Core Data Resource.

Faecal phageome of healthy individuals: presence of antibiotic resistance genes and variations caused by ciprofloxacin treatment.

OBJECTIVES: Antimicrobial resistance genes (ARGs) can be transferred by means of mobile genetic elements, which play a critical role in the dissemination of resistance in the bacterial community. ARG transmission within mobile genetic elements has been reported in plasmids and transposons but less frequently in bacteriophages. Here, the bacteriophage fraction of seven human faecal samples was purified and deep-sequenced to detect the presence of ARGs in the phage particles. METHODS: Seven faecal samples (five from healthy individuals and two from a patient before and after receiving ciprofloxacin treatment) were used to extract phage DNA, which was purified and then sequenced in a MiSeq (Illumina). Generated reads were checked for quality and assembled, and then the generated contigs analysed with Kraken, PHASTER, VirSorter and Prokka. Some genes were also validated by quantitative PCR. RESULTS: Analysis of the purified phage DNA by Kraken identified from 4 to 266 viruses in the samples. The viral fraction corresponded mainly to the order Caudovirales, including phages from the Siphoviridae and Myoviridae families. Bacterial genes associated with antimicrobial resistance were detected in the viral DNA, as confirmed by quantitative PCR. Higher densities of ARG-carrying phage particles were observed in the post- versus pre-ciprofloxacin treatment sample. CONCLUSIONS: The finding of ARGs in phage particles supports the description of phages as mobile elements contributing to the dissemination of bacterial antibiotic resistance and suggests ciprofloxacin treatment may play a role in the release of ARG-carrying particles, thereby increasing resistance.

Acinetobacter dijkshoorniae sp. nov., a member of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex mainly recovered from clinical samples in different countries.

The recent advances in bacterial species identification methods have led to the rapid taxonomic diversification of the genus Acinetobacter. In the present study, phenotypic and molecular methods have been used to determine the taxonomic position of a group of 12 genotypically distinct strains belonging to the Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) complex, initially described by Gerner-Smidt and Tjernberg in 1993, that are closely related to Acinetobacter pittii. Strains characterized in this study originated mostly from human samples obtained in different countries over a period of 15 years. rpoB gene sequences and multilocus sequence typing were used for comparisons against 94 strains representing all species included in the ACB complex. Cluster analysis based on such sequences showed that all 12 strains grouped together in a distinct clade closest to Acinetobacter pittiithat was supported by bootstrap values of 99 %. Values of average nucleotide identity based on blast between the genome sequence of strain JVAP01T (NCBI accession no. LJPG00000000) and those of other species from the ACB complex were always <91.2 %, supporting the species status of the group. In addition, the metabolic characteristics of the group matched those of the ACB complex and the analysis of their protein signatures by matrix-assisted laser desorption ionization time-of-flight MS identified some specific peaks. Our results support the designation of these strains as representing a novel species, for which the name Acinetobacter dijkshoorniae sp. nov. is proposed. The type strain is JVAP01T (=CECT 9134T=LMG 29605T).

Identification of NDM-1 in a Putatively Novel Acinetobacter Species ("NB14") Closely Related to Acinetobacter pittii.

In this study, we describe the molecular characterization of a plasmid-located blaNDM-1 harbored by an Acinetobacter clinical isolate recovered from a patient in Turkey that putatively constitutes a novel Acinetobacter species, as shown by its distinct ARDRA (amplified 16S ribosomal DNA restriction analysis) profile and molecular sequencing techniques. blaNDM-1 was carried by a conjugative plasmid widespread among non-baumannii Acinetobacter isolates, suggesting its potential for dissemination before reaching more clinically relevant Acinetobacter species.

Impact of quinolone-resistance acquisition on biofilm production and fitness in Salmonella enterica.

OBJECTIVES: To investigate the potential relationship between quinolone resistance and biofilm production in a collection of Salmonella enterica clinical isolates and in S. enterica serovar Typhimurium serial mutants with increasing resistance to ciprofloxacin. METHODS: Nalidixic acid susceptibility and biofilm formation were assessed in a collection of 122 S. enterica clinical isolates. An in vitro quinolone-resistant mutant, 59-64, was obtained from a biofilm-producing and quinolone-susceptible clinical isolate, 59-wt, in a multistep selection process after increasing ciprofloxacin concentrations. The quinolone resistance mechanisms [target gene and multidrug resistance (MDR) regulatory mutations, MICs of several antibiotics, cell envelope protein analysis, real-time PCR and ciprofloxacin accumulation] were characterized for mutant strains. In addition, analysis of fitness, biofilm formation, rdar morphotype and expression of biofilm-related genes by real-time PCR were also determined. RESULTS: Nalidixic acid-susceptible S. enterica strains were more prevalent in producing biofilm than the resistant counterparts. Strain 59-64 acquired five target gene mutations and showed an MDR phenotype. AcrAB and acrF overexpression were ruled out, whereas TolC did show increased expression in 59-64, which, in addition, accumulated less ciprofloxacin. Consistently, increased ramA expression was seen in 59-64 and attributed to a mutation within its promoter. Reduced biofilm production related to diminished csgB expression as well as reduced fitness was seen for 59-64, which was unable to form the rdar morphotype. CONCLUSIONS: Quinolone resistance acquisition may be associated with decreased production of biofilm due to lower csgB expression. Efflux, biofilm production and fitness seem to be interrelated.

Trans-ancestral genome-wide association study of longitudinal pubertal height growth and shared heritability with adult health outcomes.

BACKGROUND: Pubertal growth patterns correlate with future health outcomes. However, the genetic mechanisms mediating growth trajectories remain largely unknown. Here, we modeled longitudinal height growth with Super-Imposition by Translation And Rotation (SITAR) growth curve analysis on ~ 56,000 trans-ancestry samples with repeated height measurements from age 5 years to adulthood. We performed genetic analysis on six phenotypes representing the magnitude, timing, and intensity of the pubertal growth spurt. To investigate the lifelong impact of genetic variants associated with pubertal growth trajectories, we performed genetic correlation analyses and phenome-wide association studies in the Penn Medicine BioBank and the UK Biobank. RESULTS: Large-scale growth modeling enables an unprecedented view of adolescent growth across contemporary and 20th-century pediatric cohorts. We identify 26 genome-wide significant loci and leverage trans-ancestry data to perform fine-mapping. Our data reveals genetic relationships between pediatric height growth and health across the life course, with different growth trajectories correlated with different outcomes. For instance, a faster tempo of pubertal growth correlates with higher bone mineral density, HOMA-IR, fasting insulin, type 2 diabetes, and lung cancer, whereas being taller at early puberty, taller across puberty, and having quicker pubertal growth were associated with higher risk for atrial fibrillation. CONCLUSION: We report novel genetic associations with the tempo of pubertal growth and find that genetic determinants of growth are correlated with reproductive, glycemic, respiratory, and cardiac traits in adulthood. These results aid in identifying specific growth trajectories impacting lifelong health and show that there may not be a single "optimal" pubertal growth pattern.

Influence of FTO variants on obesity, inflammation and cardiovascular disease risk biomarkers in Spanish children: a case-control multicentre study.

BACKGROUND: Variants in the FTO gene have been associated with obesity in children, but this association has not been shown with other biomarkers. We assessed the association of 52 FTO polymorphisms, spanning the whole gene, with obesity and estimated the influence of these polymorphisms on anthropometric, clinical and metabolic parameters as well as inflammation and cardiovascular disease (CVD) risk biomarkers among Spanish children. METHODS: A multicentre case-control study was conducted in 534 children (292 obese and 242 with normal-BMI). Anthropometric, clinical, metabolic, inflammation and CVD risk markers were compared using the Student's t-test for unpaired samples. The genotype relative risk was assessed by comparing the obese and normal-BMI group, calculating the odds ratio. The association of each SNP with phenotypic parameters was analysed using either logistic or linear regression analysis. RESULTS: All anthropometric, clinical and metabolic factors as well as inflammatory and CVD risk biomarkers were higher in the obese than in the normal-BMI group, except adiponectin and HDL-c that were lower, and glucose, LDL-c, and metalloproteinase-9 that did not show difference. Four polymorphisms (rs9935401, rs9939609, rs9928094 and rs9930333) were positively associated with obesity and in linkage disequilibrium between each other; the haplotype including the risk alleles of these polymorphisms showed a high risk for obesity. The rs8061518 was negatively associated with obesity and the haplotype including this SNP and rs3826169, rs17818902 and rs7190053 showed a decreased risk for obesity. Additionally, the rs8061518 was associated with weight, diastolic blood pressure, insulin, homeostatic model assessment of insulin resistance, leptin, and active plasminogen inhibitor activator-1 after sex and age adjustment; however, after an additional BMI adjustment, this polymorphism remained associated only with leptin. CONCLUSIONS: We validated the previous reported association of genetic variability in intron 1 of the FTO gene with the risk of obesity and found no association with other related traits in this region of the gene. We have observed strong statistical evidence for an association of rs8061518 in intron 3 of the gene with decreased risk of obesity and low concentration of leptin.

Chicken liver is a potential reservoir of bacteriophages and phage-derived particles containing antibiotic resistance genes.

Poultry meat production is one of the most important agri-food industries in the world. The selective pressure exerted by widespread prophylactic or therapeutic use of antibiotics in intensive chicken farming favours the development of drug resistance in bacterial populations. Chicken liver, closely connected with the intestinal tract, has been directly involved in food-borne infections and found to be contaminated with pathogenic bacteria, including Campylobacter and Salmonella. In this study, 74 chicken livers, divided into sterile and non-sterile groups, were analysed, not only for microbial indicators but also for the presence of phages and phage particles containing antibiotic resistance genes (ARGs). Both bacteria and phages were detected in liver tissues, including those dissected under sterile conditions. The phages were able to infect Escherichia coli and showed a Siphovirus morphology. The chicken livers contained from 103 to 106 phage particles per g, which carried a range of ARGs (blaTEM , blaCTx-M-1 , sul1, qnrA, armA and tetW) detected by qPCR. The presence of phages in chicken liver, mostly infecting E. coli, was confirmed by metagenomic analysis, although this technique was not sufficiently sensitive to identify ARGs. In addition, ARG-carrying phages were detected in chicken faeces by qPCR in a previous study of the group. Comparison of the viromes of faeces and liver showed a strong coincidence of species, which suggests that the phages found in the liver originate in faeces. These findings suggests that phages, like bacteria, can translocate from the gut to the liver, which may therefore constitute a potential reservoir of antibiotic resistance genes.

Machine learning methods applied to genotyping data capture interactions between single nucleotide variants in late onset Alzheimer's disease.

Introduction: Genome-wide association studies (GWAS) in late onset Alzheimer's disease (LOAD) provide lists of individual genetic determinants. However, GWAS do not capture the synergistic effects among multiple genetic variants and lack good specificity. Methods: We applied tree-based machine learning algorithms (MLs) to discriminate LOAD (>700 individuals) and age-matched unaffected subjects in UK Biobank with single nucleotide variants (SNVs) from Alzheimer's disease (AD) studies, obtaining specific genomic profiles with the prioritized SNVs. Results: MLs prioritized a set of SNVs located in genes PVRL2, TOMM40, APOE, and APOC1, also influencing gene expression and splicing. The genomic profiles in this region showed interaction patterns involving rs405509 and rs1160985, also present in the Alzheimer's Disease Neuroimaging Initiative (ADNI) dataset. rs405509 located in APOE promoter interacts with rs429358 among others, seemingly neutralizing their predisposing effect. Discussion: Our approach efficiently discriminates LOAD from controls, capturing genomic profiles defined by interactions among SNVs in a hot-spot region.

NGS-based liquid biopsy profiling identifies mechanisms of resistance to ALK inhibitors: a step toward personalized NSCLC treatment.

Despite impressive and durable responses, nonsmall cell lung cancer (NSCLC) patients treated with anaplastic lymphoma kinase (ALK) inhibitors (ALK-Is) ultimately progress due to development of resistance. Here, we have evaluated the clinical utility of circulating tumor DNA (ctDNA) profiling by next-generation sequencing (NGS) upon disease progression. We collected 26 plasma and two cerebrospinal fluid samples from 24 advanced ALK-positive NSCLC patients at disease progression to an ALK-I. These samples were analyzed by NGS and digital PCR. A tool to retrieve variants at the ALK locus was developed (VALK tool). We identified at least one resistance mutation in the ALK locus in ten (38.5%) plasma samples; the G1269A and G1202R mutations were the most prevalent among patients progressing to first- and second-generation ALK-Is, respectively. Overall, 61 somatic mutations were detected in 14 genes: TP53, ALK, PIK3CA, SMAD4, MAP2K1 (MEK1), FGFR2, FGFR3, BRAF, EGFR, IDH2, MYC, MET, CCND3, and CCND1. Specifically, a deletion in exon 19 in EGFR, a non-V600 BRAF mutation (G466V), and the F129L mutation in MAP2K1 were identified in four patients who showed no objective survival benefit from ALK-Is. Potential ALK-I-resistance mutations were also found in PIK3CA and IDH2. Finally, a c-MYC gain, along with a loss of CCND1 and FGFR3, was detected in a patient progressing on a first-line treatment with crizotinib. We conclude that NGS analysis of liquid biopsies upon disease progression identified different putative ALK-I-resistance mutations in most cases and could be a valuable approach for therapy decision making.

Rare variant analysis in eczema identifies exonic variants in DUSP1, NOTCH4 and SLC9A4.

Previous genome-wide association studies revealed multiple common variants involved in eczema but the role of rare variants remains to be elucidated. Here, we investigate the role of rare variants in eczema susceptibility. We meta-analyze 21 study populations including 20,016 eczema cases and 380,433 controls. Rare variants are imputed with high accuracy using large population-based reference panels. We identify rare exonic variants in DUSP1, NOTCH4, and SLC9A4 to be associated with eczema. In DUSP1 and NOTCH4 missense variants are predicted to impact conserved functional domains. In addition, five novel common variants at SATB1-AS1/KCNH8, TRIB1/LINC00861, ZBTB1, TBX21/OSBPL7, and CSF2RB are discovered. While genes prioritized based on rare variants are significantly up-regulated in the skin, common variants point to immune cell function. Over 20% of the single nucleotide variant-based heritability is attributable to rare and low-frequency variants. The identified rare/low-frequency variants located in functional protein domains point to promising targets for novel therapeutic approaches to eczema.

Unravelling the consequences of the bacteriophages in human samples.

Bacteriophages are abundant in human biomes and therefore in human clinical samples. Although this is usually not considered, they might interfere with the recovery of bacterial pathogens at two levels: 1) by propagating in the enrichment cultures used to isolate the infectious agent, causing the lysis of the bacterial host and 2) by the detection of bacterial genes inside the phage capsids that mislead the presence of the bacterial pathogen. To unravel these interferences, human samples (n = 271) were analyzed and infectious phages were observed in 11% of blood culture, 28% of serum, 45% of ascitic fluid, 14% of cerebrospinal fluid and 23% of urine samples. The genetic content of phage particles from a pool of urine and ascitic fluid samples corresponded to bacteriophages infecting different bacterial genera. In addition, many bacterial genes packaged in the phage capsids, including antibiotic resistance genes and 16S rRNA genes, were detected in the viromes. Phage interference can be minimized applying a simple procedure that reduced the content of phages up to 3 logs while maintaining the bacterial load. This method reduced the detection of phage genes avoiding the interference with molecular detection of bacteria and reduced the phage propagation in the cultures, enhancing the recovery of bacteria up to 6 logs.

The use of gene array technology and proteomics in the search of new targets of diseases for therapeutics.

The advent of functional genomics has been greatly broadening our view and accelerating our way in numerous medical research fields. The complete genomic data acquired from the human genome project and the desperate clinical need of comprehensive analytical tools to study complex diseases, has allowed rapid evolution of genomic and proteomic technologies, speeding the rate and number of discoveries in new biomarkers. By jointly using genomics, proteomics and bioinformatics there is a great potential to make considerable contribution to biomarker identification and to revolutionize both the development of new therapies and drug development process.

Low-frequency variation in TP53 has large effects on head circumference and intracranial volume.

Cranial growth and development is a complex process which affects the closely related traits of head circumference (HC) and intracranial volume (ICV). The underlying genetic influences shaping these traits during the transition from childhood to adulthood are little understood, but might include both age-specific genetic factors and low-frequency genetic variation. Here, we model the developmental genetic architecture of HC, showing this is genetically stable and correlated with genetic determinants of ICV. Investigating up to 46,000 children and adults of European descent, we identify association with final HC and/or final ICV + HC at 9 novel common and low-frequency loci, illustrating that genetic variation from a wide allele frequency spectrum contributes to cranial growth. The largest effects are reported for low-frequency variants within TP53, with 0.5 cm wider heads in increaser-allele carriers versus non-carriers during mid-childhood, suggesting a previously unrecognized role of TP53 transcripts in human cranial development.

Circulating miRNAs, isomiRs and small RNA clusters in human plasma and breast milk.

Circulating small RNAs, including miRNAs but also isomiRs and other RNA species, have the potential to be used as non-invasive biomarkers for communicable and non-communicable diseases. This study aims to characterize and compare small RNA profiles in human biofluids. For this purpose, RNA was extracted from plasma and breast milk samples from 15 healthy postpartum mothers. Small RNA libraries were prepared with the NEBNext® small RNA library preparation kit and sequenced in an Illumina HiSeq2000 platform. miRNAs, isomiRs and clusters of small RNAs were annotated using seqBuster/seqCluster framework in 5 plasma and 10 milk samples that passed the initial quality control. The RNA yield was 81 ng/mL [standard deviation (SD): 41] and 3985 ng/mL (SD: 3767) for plasma and breast milk, respectively. Mean number of good quality reads was 4.04 million (M) (40.01% of the reads) in plasma and 12.5M (89.6%) in breast milk. One thousand one hundred eighty two miRNAs, 12,084 isomiRs and 1,053 small RNA clusters that included piwi-interfering RNAs (piRNAs), tRNAs, small nucleolar RNAs (snoRNA) and small nuclear RNAs (snRNAs) were detected. Samples grouped by biofluid, with 308 miRNAs, 1,790 isomiRs and 778 small RNA clusters differentially detected. In summary, plasma and milk showed a different small RNA profile. In both, miRNAs, piRNAs, tRNAs, snRNAs, and snoRNAs were identified, confirming the presence of non-miRNA species in plasma, and describing them for the first time in milk.

Characterization of Plasmodium vivax Proteins in Plasma-Derived Exosomes From Malaria-Infected Liver-Chimeric Humanized Mice.

Exosomes are extracellular vesicles of endocytic origin containing molecular signatures implying the cell of origin; thus, they offer a unique opportunity to discover biomarkers of disease. Plasmodium vivax, responsible for more than half of all malaria cases outside Africa, is a major obstacle in the goal of malaria elimination due to the presence of dormant liver stages (hypnozoites), which after the initial infection may reactivate to cause disease. Hypnozoite infection is asymptomatic and there are currently no diagnostic tools to detect their presence. The human liver-chimeric (FRG huHep) mouse is a robust P. vivax infection model for exo-erythrocytic development of liver stages, including hypnozoites. We studied the proteome of plasma-derived exosomes isolated from P. vivax infected FRG huHep mice with the objective of identifying liver-stage expressed parasite proteins indicative of infection. Proteomic analysis of these exosomes showed the presence of 290 and 234 proteins from mouse and human origin, respectively, including canonical exosomal markers. Human proteins include proteins previously detected in liver-derived exosomes, highlighting the potential of this chimeric mouse model to study plasma exosomes derived unequivocally from human hepatocytes. Noticeably, we identified 17 parasite proteins including enzymes, surface proteins, components of the endocytic pathway and translation machinery, as well as uncharacterized proteins. Western blot analysis validated the presence of human arginase-I and an uncharacterized P. vivax protein in plasma-derived exosomes. This study represents a proof-of-principle that plasma-derived exosomes from P. vivax infected FRG-huHep mice contain human hepatocyte and P. vivax proteins with the potential to unveil biological features of liver infection and identify biomarkers of hypnozoite infection.

Draft Genome Sequence of JVAP01T, the Type Strain of the Novel Species Acinetobacter dijkshoorniae.

Here, we report the draft genome sequence of the type strain of Acinetobacter dijkshoorniae, a novel human pathogen within the Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) complex. Strain JVAP01T has an estimated genome size of 3.9 Mb, exhibits a 38.8% G+C content, and carries a plasmid with the blaNDM-1 carbapenemase gene.

First insights into the pleiotropic role of vrf (yedF), a newly characterized gene of Salmonella Typhimurium.

Salmonella possesses virulence determinants that allow replication under extreme conditions and invasion of host cells, causing disease. Here, we examined four putative genes predicted to encode membrane proteins (ydiY, ybdJ, STM1441 and ynaJ) and a putative transcriptional factor (yedF). These genes were identified in a previous study of a S. Typhimurium clinical isolate and its multidrug-resistant counterpart. For STM1441 and yedF a reduced ability to interact with HeLa cells was observed in the knock-out mutants, but an increase in this ability was absent when these genes were overexpressed, except for yedF which phenotype was rescued when yedF was restored. In the absence of yedF, decreased expression was seen for: i) virulence-related genes involved in motility, chemotaxis, attachment and survival inside the host cell; ii) global regulators of the invasion process (hilA, hilC and hilD); and iii) factors involved in LPS biosynthesis. In contrast, an increased expression was observed for anaerobic metabolism genes. We propose yedF is involved in the regulation of Salmonella pathogenesis and contributes to the activation of the virulence machinery. Moreover, we propose that, when oxygen is available, yedF contributes sustained repression of the anaerobic pathway. Therefore, we recommend this gene be named vrf, for virulence-related factor.