The gold-standard COVID-19 diagnosis relies on detecting SARS-CoV-2 using RNA purification and one-step retrotranscription and quantitative PCR (RT-qPCR). Based on the urgent need for high-throughput screening, we tested the performance of three alternative, simple and affordable protocols to rapidly detect SARS-CoV-2, bypassing the long and tedious RNA extraction step and reducing the time to viral detection. We evaluated three methods based on direct nasopharyngeal swab viral transmission medium (VTM) heating before the RT-qPCR: a) direct without additives; b) in a formamide-EDTA (FAE) buffer, c) in a RNAsnapTM buffer. Although with a delay in cycle threshold compared to the gold-standard, we found consistent results in nasopharyngeal swab samples that were subject to a direct 70°C incubation for 10 min. Our findings provide valuable options to overcome any supply chain issue and help to increase the throughput of diagnostic tests, thereby complementing standard diagnosis.

The transactive response DNA-binding protein (TARDBP/TDP-43) influences the processing of diverse transcripts, including that of histone deacetylase 6 (HDAC6). Here, we assessed TDP-43 activity in terms of regulating CD4+ T-cell permissivity to HIV-1 infection. We observed that overexpression of wt-TDP-43 increased both mRNA and protein levels of HDAC6, resulting in impaired HIV-1 infection independently of the viral envelope glycoprotein complex (Env) tropism. Consistently, using an HIV-1 Env-mediated cell-to-cell fusion model, the overexpression of TDP-43 levels negatively affected viral Env fusion capacity. Silencing of endogenous TDP-43 significantly decreased HDAC6 levels and increased the fusogenic and infection activities of the HIV-1 Env. Using pseudovirus bearing primary viral Envs from HIV-1 individuals, overexpression of wt-TDP-43 strongly reduced the infection activity of Envs from viremic non-progressors (VNP) and rapid progressors (RP) patients down to the levels of the inefficient HIV-1 Envs observed in long-term non-progressor elite controllers (LTNP-EC). On the contrary, silencing endogenous TDP-43 significantly favored the infectivity of primary Envs from VNP and RP individuals, and notably increased the infection of those from LTNP-EC. Taken together, our results indicate that TDP-43 shapes cell permissivity to HIV-1 infection, affecting viral Env fusion and infection capacities by altering the HDAC6 levels and associated tubulin-deacetylase anti-HIV-1 activity.

Whole-exome sequencing has become a popular technique in research and clinical settings, assisting in disease diagnosis and increasing the understanding of disease pathogenesis. In this study, we aimed to compare common enrichment capture solutions available in the market. Peripheral blood-purified DNA samples were enriched with SureSelect^QXT V6 (Agilent) and various Illumina solutions: TruSeq DNA Nano, TruSeq DNA Exome, Nextera DNA Exome, and Illumina DNA Prep with Enrichment, and sequenced on a HiSeq 4000. We found that their percentage of duplicate reads was as much as 2 times higher than previously reported values for the previous HiSeq series. SureSelect^QXT and Illumina DNA Prep with Enrichment showed the best average on-target coverage, which improved when off-target regions were included. At high coverage levels and in shared bases, these two solutions and TruSeq DNA Exome provided three of the best performances. With respect to the number of small variants detected, SureSelect^QXT presented the lowest number of detected variants in target regions. When off-target regions were considered, its ability equalized to other solutions. Our results show SureSelect^QXT and Illumina DNA Prep with Enrichment to be the best enrichment capture solutions.

Background: Hereditary angioedema is a rare genetic condition caused by C1 esterase inhibitor deficiency, dysfunction, or kinin cascade dysregulation, leading to an increased bradykinin plasma concentration. Hereditary angioedema is a poorly recognized clinical entity and is very often misdiagnosed as a histaminergic angioedema. Despite its genetic nature, first-line genetic screening is not integrated in routine diagnosis. Consequently, a delay in the diagnosis, and inaccurate or incomplete diagnosis and treatment of hereditary angioedema are common. Objective: In agreement with recent recommendations from the International Consensus on the Use of Genetics in the Management of Hereditary Angioedema, to facilitate the clinical diagnosis and adapt it to the paradigm of precision medicine and next-generation sequencing–based genetic tests, we aimed to develop a genetic annotation tool, termed Hereditary Angioedema Database Annotation (HADA). Methods: HADA is built on top of a database of known variants affecting function, including precomputed pathogenic assessment of each variant and a ranked classification according to the current guidelines from the American College of Medical Genetics and Genomics. Results: HADA is provided as a freely accessible, user-friendly web-based interface with versatility for the entry of genetic information. The underlying database can also be incorporated into automated command-line stand-alone annotation tools. Conclusions: HADA can achieve the rapid detection of variants affecting function for different hereditary angioedema types, and further integrates useful information to reduce the diagnosis odyssey and improve its delay.

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.

BACKGROUND Hereditary angioedema is a rare genetic condition caused by C1 esterase inhibitor deficiency, dysfunction, or kinin cascade dysregulation, leading to an increased bradykinin plasma concentration. Hereditary angioedema is a poorly recognized clinical entity and is very often misdiagnosed as a histaminergic angioedema. Despite its genetic nature, first-line genetic screening is not integrated in routine diagnosis. Consequently, a delay in the diagnosis, and inaccurate or incomplete diagnosis and treatment of hereditary angioedema are common. OBJECTIVE In agreement with recent recommendations from the International Consensus on the Use of Genetics in the Management of Hereditary Angioedema, to facilitate the clinical diagnosis and adapt it to the paradigm of precision medicine and next-generation sequencing–based genetic tests, we aimed to develop a genetic annotation tool, termed Hereditary Angioedema Database Annotation (HADA). METHODS HADA is built on top of a database of known variants affecting function, including precomputed pathogenic assessment of each variant and a ranked classification according to the current guidelines from the American College of Medical Genetics and Genomics. RESULTS HADA is provided as a freely accessible, user-friendly web-based interface with versatility for the entry of genetic information. The underlying database can also be incorporated into automated command-line stand-alone annotation tools. CONCLUSIONS HADA can achieve the rapid detection of variants affecting function for different hereditary angioedema types, and further integrates useful information to reduce the diagnosis odyssey and improve its delay.