Evaluation of a whole-exome sequencing pipeline and benchmarking of causal germline variant prioritizers.

Most causal variants of Mendelian diseases are exonic. Whole-exome sequencing (WES) has become the diagnostic gold standard, but causative variant prioritization constitutes a bottleneck. Here we assessed an in-house sample-to-sequence pipeline and benchmarked free prioritization tools for germline causal variants from WES data. WES of 61 unselected patients with a known genetic disease cause was obtained. Variant prioritizations were performed by diverse tools and recorded to obtain a diagnostic yield when the causal variant was present in the first, fifth, and 10th top rankings. A fraction of causal variants was not captured by WES (8.2%) or did not pass the quality control criteria (13.1%). Most of the applications inspected were unavailable or had technical limitations, leaving nine tools for complete evaluation. Exomiser performed best in the top first rankings, while LIRICAL led in the top fifth rankings. Based on the more conservative top 10th rankings, Xrare had the highest diagnostic yield, followed by a three-way tie among Exomiser, LIRICAL, and PhenIX, then followed by AMELIE, TAPES, Phen-Gen,  AIVar, and VarNote-PAT. Xrare, Exomiser, LIRICAL, and PhenIX are the most efficient options for variant prioritization in real patient WES data.

Transactive Response DNA-Binding Protein (TARDBP/TDP-43) Regulates Cell Permissivity to HIV-1 Infection by Acting on HDAC6.

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.

Interactive Web-Based Resource for Annotation of Genetic Variants Causing Hereditary Angioedema (HADA): Database Development, Implementation, and Validation.

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.

Benchmarking of human Y-chromosomal haplogroup classifiers with whole-genome and whole-exome sequence data.

In anthropological, medical, and forensic studies, the nonrecombinant region of the human Y chromosome (NRY) enables accurate reconstruction of pedigree relationships and retrieval of ancestral information. Using high-throughput sequencing (HTS) data, we present a benchmarking analysis of command-line tools for NRY haplogroup classification. The evaluation was performed using paired Illumina data from whole-genome sequencing (WGS) and whole-exome sequencing (WES) experiments from 50 unrelated donors. Additionally, as a validation, we also used paired WGS/WES datasets of 54 individuals from the 1000 Genomes Project. Finally, we evaluated the tools on data from third-generation HTS obtained from a subset of donors and one reference sample. Our results show that WES, despite typically offering less genealogical resolution than WGS, is an effective method for determining the NRY haplogroup. Y-LineageTracker and Yleaf showed the highest accuracy for WGS data, classifying precisely 98% and 96% of the samples, respectively. Yleaf outperforms all benchmarked tools in the WES data, classifying approximately 90% of the samples. Yleaf, Y-LineageTracker, and pathPhynder can correctly classify most samples (88%) sequenced with third-generation HTS. As a result, Yleaf provides the best performance for applications that use WGS and WES. Overall, our study offers researchers with a guide that allows them to select the most appropriate tool to analyze the NRY region using both second- and third-generation HTS data.

Reinfection rate and disease severity of the BA.5 Omicron SARS-CoV-2 lineage compared to previously circulating variants of concern in the Canary Islands (Spain).

ABSTRACTThe emergence of the Omicron SARS-CoV-2 variant of concern has changed the COVID-19 scenario as this variant is characterized by high transmissibility and immune evasion ability. To evaluate the impact of this variant on the Canary Islands (Spain) population, we determined the reinfection rates and disease severity associated with the Omicron sublineages and the previously circulating variants of concern. We performed a retrospective observational study on 21,745 SARS-CoV-2 viral genomes collected from December 2020 to July 2022 in the Canary Islands (Spain). We compared the reinfection rates between lineages using pairwise proportion and Fisher's exact tests. To assess disease severity, we studied the association of Alpha, Delta, BA.1, BA.2, BA.5, and other risk factors on 28-day hospital mortality using logistic regression and Cox proportional hazard models. We observed 127 bona fide reinfection cases throughout the study period. We found that BA.5 had the highest reinfection rate compared to other lineages (vs. Delta p = 2.89 × 10-25; vs. BA.1 p = 5.17 × 10-11; vs. BA.2 p = 0.002). Among the 1,094 hospitalized patients, multivariate logistic regression showed that Alpha (Odds Ratio [OR] = 0.45, 95% Confidence Interval [CI] = 0.23-0.87, p = 0.02), BA.2 (OR = 0.38, 95% CI = 0.22-0.63, p = 1.91 × 10-4), and BA.5 (OR = 0.30, 95% CI = 0.16-0.55, p = 1.05 × 10-4) had lower 28-day hospital mortality compared to Delta. These results were confirmed by using Cox proportional hazard models. Omicron lineages, and in particular BA.5, were associated with higher reinfection rates and lower disease severity (28-day hospital mortality) than previously circulating variants of concern.

Digging into the admixture strata of current-day Canary Islanders based on mitogenomes.

The conquest of the Canary Islands by Europeans began at the beginning of the 15th century and culminated in 1496 with the surrender of the aborigines. The collapse of the aboriginal population during the conquest and the arrival of settlers caused a drastic change in the demographic composition of the archipelago. To shed light on this historical process, we analyzed 896 mitogenomes of current inhabitants from the seven main islands. Our findings confirm the continuity of aboriginal maternal contributions and the persistence of their genetic footprints in the current population, even at higher levels (>60% on average) than previously evidenced. Moreover, the age estimates for most autochthonous founder lineages support a first aboriginal arrival to the islands at the beginning of the first millennium. We also revealed for the first time that the main recognizable genetic influences from Europe are from Portuguese and Galicians.

Bioinformatic approaches to draft the viral genome sequence of Canary Islands cases related to the multicountry mpox virus 2022-outbreak.

On July 23, 2022, monkeypox disease (mpox) was declared a Public Emergency of International Concern (PHEIC) by the World Health Organization (WHO) due to a multicountry outbreak. In Europe, several cases of mpox virus (MPXV) infection related to this outbreak were detected in the Canary Islands (Spain). Here we describe the combination of viral DNA sequencing and bioinformatic approaches, including methods for de novo genome assembly and short- and long-read technologies, used to reconstruct the first MPXV genome isolated in the Canary Islands on the 31st of May 2022 from a male adult patient with mild symptoms. The same sequencing and bioinformatic approaches were then validated with three other positive cases of MPXV infection from the same mpox outbreak. We obtained the best results using a reference-based approach with short reads, evidencing 46-79 nucleotide variants against viral sequences from the 2018-2019 mpox outbreak and placing the viral sequences in the new B.1 sublineage of clade IIb of the MPXV classification. This study of MPXV demonstrates the potential of metagenomics sequencing for rapid and precise pathogen identification.

Tracing the trajectories of SARS-CoV-2 variants of concern between December 2020 and September 2021 in the Canary Islands (Spain).

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 sublineages) 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.

Developing CIRdb as a catalog of natural genetic variation in the Canary Islanders.

The current inhabitants of the Canary Islands have a unique genetic makeup in the European diversity landscape due to the existence of African footprints from recent admixture events, especially of North African components (> 20%). The underrepresentation of non-Europeans in genetic studies and the sizable North African ancestry, which is nearly absent from all existing catalogs of worldwide genetic diversity, justify the need to develop CIRdb, a population-specific reference catalog of natural genetic variation in the Canary Islanders. Based on array genotyping of the selected unrelated donors and comparisons against available datasets from European, sub-Saharan, and North African populations, we illustrate the intermediate genetic differentiation of Canary Islanders between Europeans and North Africans and the existence of within-population differences that are likely driven by genetic isolation. Here we describe the overall design and the methods that are being implemented to further develop CIRdb. This resource will help to strengthen the implementation of Precision Medicine in this population by contributing to increase the diversity in genetic studies. Among others, this will translate into improved ability to fine map disease genes and simplify the identification of causal variants and estimate the prevalence of unattended Mendelian diseases.

A catalog of the genetic causes of hereditary angioedema in the Canary Islands (Spain).

Hereditary angioedema (HAE) is a rare disease where known causes involve C1 inhibitor dysfunction or dysregulation of the kinin cascade. The updated HAE management guidelines recommend performing genetic tests to reach a precise diagnosis. Unfortunately, genetic tests are still uncommon in the diagnosis routine. Here, we characterized for the first time the genetic causes of HAE in affected families from the Canary Islands (Spain). Whole-exome sequencing data was obtained from 41 affected patients and unaffected relatives from 29 unrelated families identified in the archipelago. The Hereditary Angioedema Database Annotation (HADA) tool was used for pathogenicity classification and causal variant prioritization among the genes known to cause HAE. Manual reclassification of prioritized variants was used in those families lacking known causal variants. We detected a total of eight different variants causing HAE in this patient series, affecting essentially SERPING1 and F12 genes, one of them being a novel SERPING1 variant (c.686-12A>G) with a predicted splicing effect which was reclassified as likely pathogenic in one family. Altogether, the diagnostic yield by assessing previously reported causal genes and considering variant reclassifications according to the American College of Medical Genetics guidelines reached 66.7% (95% Confidence Interval [CI]: 30.1-91.0) in families with more than one affected member and 10.0% (95% CI: 1.8-33.1) among cases without family information for the disease. Despite the genetic causes of many patients remain to be identified, our results reinforce the need of genetic tests as first-tier diagnostic tool in this disease, as recommended by the international WAO/EAACI guidelines for the management of HAE.

A benchmarking of human mitochondrial DNA haplogroup classifiers from whole-genome and whole-exome sequence data.

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. We also assessed 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.

Fast SARS-CoV-2 detection by RT-qPCR in preheated nasopharyngeal swab samples.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Our findings provide valuable options to overcome any supply chain issue and help to increase the throughput of diagnostic tests, thereby complementing standard diagnosis.

Evaluation of Whole-Exome Enrichment Solutions: Lessons from the High-End of the Short-Read Sequencing Scale.

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 SureSelectQXT 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. SureSelectQXT 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, SureSelectQXT 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 SureSelectQXT and Illumina DNA Prep with Enrichment to be the best enrichment capture solutions.