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2.AbstractMethicillin-resistantStaphylococcus aureus(MRSA) is one of the major human pathogens, causing a wide range of infections from food poisoning to necrotizing pneumonia, endocarditis, or septicemia. It could carry numerous resistance genes and virulence factors, some of which are related to the severity of the infection, being also regarded as a potential “Super Bug”. In Paraguay, the prevalence of CC30-ST30-IV clones is the leading cause ofS. aureusinfections both at the regional level and in pediatric population. Here we aimed to deeply analyze the genomic features of MRSA isolates that cause invasive infections in Paraguayan children. An observational, descriptive, cross-sectional study was designed to analyze representative MRSA isolates of the main clones identified between 2009 to 2013 in Paraguayan children. All the genetically linked MRSA isolates were recovered from diverse clinical sources, patients, and hospitals at broad gap periods. Cases were primarily community-acquired, which excludes in-hospital transmission or outbreaks. MRSA isolates were analyzed with short-read paired-end sequencing and assessed for the virulome, resistome, and phylogenetic relationships. The pan-genomic analysis of these clones revealed three major and different clonal complexes (CC8, CC30, and CC5), each composed of clones closely related to each other, despite having different spa types. Furthermore, multiple virulence and resistance genes were identified for the first time in this study, pointing out the complex virulence profiles of MRSA circulating in the country. This study opens a wide range of new possibilities for future projects and trials to improve the existing knowledge on the epidemiology of MRSA circulating in Paraguay.3.Impact statementThe increasing prevalence of methicillin-resistantStaphylococcus aureus(MRSA) is a public health problem worldwide. The most frequent MRSA clones identified in Paraguay in previous studies were (including community and hospital-acquired) the Pediatric (CC5-ST5-IV), the Cordobes-Chilean (CC5-ST5-I), the SouthWest Pacific (CC30-ST30-IV) and the Brazilian (CC8-ST239-III) clones. In this study, the pan-genomic analysis of the most representative MRSA clones circulating in invasive infection in Paraguayan children over ten years (2009-2019), such as the CC30-ST30-IV, CC5-ST5-IV, and CC8-ST8-IV, were carried out to evaluate their genetic diversity, their virulence factors repertoire, and antimicrobial resistance mechanisms revealed multiple virulences and resistance genes pointing out the complex virulence profiles of MRSA circulating in Paraguay. Our work is the first genomic study of MRSA in Paraguay and will contribute to the development of genomic surveillance in the region and our understanding of this pathogen’s global epidemiology.4.Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

The non-recombinant region of the Y chromosome (NRY) contains a great number of polymorphic markers that allows to accurately reconstruct pedigree relationships and retrieve ancestral information from study samples. The analysis of NRY is typically implemented in anthropological, medical, and forensic studies. High-throughput sequencing (HTS) has profoundly increased the identification of genetic markers in the NRY genealogy and has prompted the development of automated NRY haplogroup classification tools. Here, we present a benchmarking study of five command-line tools for NRY haplogroup classification. The evaluation was done using empirical short-read HTS data from 50 unrelated donors using paired data from whole-genome sequencing (WGS) and whole-exome sequencing (WES) experiments. Besides, we evaluate the performance of the top-ranked tool in the classification of data of third generation HTS obtained from a subset of donors. Our findings demonstrate that WES can be an efficient approach to infer the NRY haplogroup, albeit generally providing a lower level of genealogical resolution than that recovered by WGS. Among the tools evaluated, YLeaf offers the best performance for both WGS and WES applications. Finally, we demonstrate that YLeaf is able to correctly classify all samples sequenced with nanopore technology from long noisy reads.

Several variants of concern (VOCs) explain most of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) epidemic waves in Europe. We aimed to dissect the spread of the SARS-CoV-2 VOCs in the Canary Islands (Spain) between December 2020 and September 2021 at a micro-geographical level. We sequenced the viral genome of 8,224 respiratory samples collected in the archipelago. We observed that Alpha (B.1.1.7) and Delta (B.1.617.2 and sub-lineages) were ubiquitously present in the islands, while Beta (B.1.351) and Gamma (P.1/P.1.1) had a heterogeneous distribution and were responsible for fewer and more controlled outbreaks. This work represents the largest effort for viral genomic surveillance in the Canary Islands so far, helping the public health bodies in decision-making throughout the pandemic.

The transactive response DNA-binding protein (TDP-43) is an important regulator of mRNA, being reported to stabilize the anti-HIV factor, histone deacetylase 6 (HDAC6). However, little is known about the role of TDP-43 in HIV infection. In this work, we seek for the TDP-43 function on regulating CD4+ T cell permissibility to HIV infection. We observed that over-expression of wt-TDP-43 in CD4+ T cells stabilized HDAC6, increasing mRNA and the protein levels of this antiviral enzyme. Under this experimental condition, HIV-1 infection was impaired, independently of the viral envelope glycoprotein (Env) complex tropism. The results obtained by using an HIV-1 Env-mediated cell-to-cell fusion model, under the same experimental conditions, suggest that the increase in TDP-43 levels negatively affects the viral Env fusion capacity. Moreover, the specific siRNA silencing of endogenous TDP-43 in target cells lead to a significant decrease in the levels of HDAC6 which consistently induces an increase in the fusogenic and infection activities of the HIV-1 Env. These observations were confirmed by using primary viral Envs from HIV+ individuals with different clinical phenotypes. An increase in the level of expression of wt-TDP-43 strongly reduced the Envs infection activity of viremic non-progressors (VNP) and rapid progressors (RP) HIV+ individuals down to the levels of the inefficient HIV-1 Envs from long-term non-progressor elite controllers (LTNP-EC) individuals. On the contrary, low levels of endogenous TDP-43, obtained after specific siRNA-TDP-43 knocking-down, significantly favors the infection activity of primary HIV-1 Envs of VNP and RP individuals, leading to an increase in the infection ability of the primary HIV-1/LTNP-EC Envs. Based on this evidence, we interpret that TDP-43 conditions cell permissibility to HIV infection by affecting viral Env fusion and infection capacities, at least by altering the cellular levels of the antiviral enzyme HDAC6.

Starting in December 2020, a sharp increase of COVID-19 cases occurred in Tenerife compared to the rest of the Canary Islands (Spain). Because of the direct touristic connections between Tenerife and the UK, and the rapid transmission and dominance of the SARS-CoV-2 B.1.1.7 variant of concern (VOC-202012/01) by the end of November 2020 in South England, here we measured the proportion of B.1.1.7 cases occurring between the 18th of December 2020 and the 25th of February 2021. Out of the 2,091 COVID-19 positive nasopharyngeal swab samples assessed, 226 showed a spike gene target failure (SGTF). Subsequent viral genome sequencing further confirmed that 93.2% of them corresponded to the B.1.1.7 lineage. Furthermore, a rapid increase in the proportion of SGTF variants was detected in up to 10.7% of positive cases during the Christmas season despite stricter measures for containing the transmission were imposed in Tenerife in the period. These results support the local transmission of SARS-CoV-2 B.1.1.7 lineage in Tenerife since late December 2020 although it is not yet dominant.

The mitochondrial genome (mtDNA) is of interest for a range of fields including evolutionary, forensic, and medical genetics. Human mitogenomes can be classified into evolutionary related haplogroups that provide ancestral information and pedigree relationships. Because of this and the advent of high-throughput sequencing (HTS) technology, there is a diversity of bioinformatic tools for haplogroup classification. We present a benchmarking of the 11 most salient tools for human mtDNA classification using empirical whole-genome (WGS) and whole-exome (WES) short-read sequencing data from 36 unrelated donors. Besides, because of its relevance, we also assess the best performing tool in third-generation long noisy read WGS data obtained with nanopore technology for a subset of the donors. We found that, for short-read WGS, most of the tools exhibit high accuracy for haplogroup classification irrespective of the input file used for the analysis. However, for short-read WES, Haplocheck and MixEmt were the most accurate tools. Based on the performance shown for WGS and WES, and the accompanying qualitative assessment, Haplocheck stands out as the most complete tool. For third-generation HTS data, we also showed that Haplocheck was able to accurately retrieve mtDNA haplogroups for all samples assessed, although only after following assembly-based approaches (either based on a referenced-based assembly or a hybrid de novo assembly). Taken together, our results provide guidance for researchers to select the most suitable tool to conduct the mtDNA analyses from HTS data.

ObjectivesLimited testing capacity has characterized the ongoing COVID-19 pandemic in Spain, hampering a timely control of outbreaks and the possibilities to reduce the escalation of community transmissions. Here we investigated the potential of using pooling of samples followed by one-step retrotranscription and quantitative PCR (RT-qPCR) to increase SARS-CoV-2 testing capacity.MethodsWe first evaluated different sample pooling (1:5, 1:10 and 1:15) prior to RNA extractions followed by standard RT-qPCR for SARS-CoV-2/COVID-19 diagnosis. The pool size achieving reproducible results in independent tests was then used for assessing nasopharyngeal samples in a tertiary hospital during August 2020.ResultsWe found that pool size of five samples achieved the highest sensitivity compared to pool sizes of 10 and 15, showing a mean (± SD) Ct shift of 3.5 ± 2.2 between the pooled test and positive samples in the pool. We then used a pool size of five to test a total of 895 pools (4,475 prospective samples) using two different RT-qPCR kits available at that time. The Real Accurate Quadruplex corona-plus PCR Kit (PathoFinder) reported the lowest mean Ct (± SD) shift (2.2 ± 2.4) among the pool and the individual samples. The strategy allows detecting individual samples in the positive pools with Cts in the range of 16.7-39.4.ConclusionsWe found that pools of five samples combined with RT-qPCR solutions helped to increase SARS-CoV-2 testing capacity with minimal loss of sensitivity compared to that resulting from testing the samples independently.

The current reference for COVID-19 diagnosis is based on the detection of SARS-CoV-2 on RNA extracts using one-step retrotranscription and quantitative PCR (RT-qPCR). Based on the urgent need for high-throughput COVID-19 screening, we tested the performance of three alternative, simple and affordable protocols to rapidly detect SARS-CoV-2, overcoming the long and tedious RNA extraction step. Although with an average increase of 6.1 (± 1.6) cycles compared to standard tests with RNA extracts, we show that RT-qPCR yielded consistent results in nasopharyngeal swab samples that were subject to a direct 70°C incubation for 10 min. Our findings provide viable options to overcome any supply chain issue and help to increase the throughput of diagnostic tests by using any qPCR device, thereby complementing standard COVID-19 testing.

Despite the reduced read length, the so-called Next-Generation Sequencing (NGS) of second-generation has allowed rapid and complete genome characterization of many species. MinION (Oxford Nanopore Technologies), a portable third-generation NGS device, enables sequencing of long DNA fragments at low cost. Here we used a low-coverage MinION sequencing in combination with short-read NGS to improve genome assembly. We tested this possibility by using MinION R9.0 with Rapid 1D kit and MiSeq with >300X paired-end 300 bp reads (Illumina, Inc.) for the genome assembly of a Streptococcus agalactiae clinical isolate (2.2 Mb). With as few as 1,171 MinION reads that covered the genome at 2.4X (the longest read being 186 Kb long), the hybrid assembly combining MinION and Illumina reads increased the N50 by 4.9-fold compared to the assembly using Illumina data alone. Almost 50% of the genome was represented into a single contig (1.02 Mb). Besides, this allowed the full reconstruction of mobile elements, including a plasmid, and improved gene annotation. Taken together, our results support that shallow MinION sequencing combined with high-throughput second-generation NGS constitutes a cost-efficient strategy for the assembly of whole genomes.