Leveraging whole-genome sequencing to estimate telomere length in plants.

Changes in telomere length are increasingly used to indicate species' response to environmental stress across diverse taxa. Despite this broad use, few studies have explored telomere length in plants. Thus, evaluation of new approaches for measuring telomeres in plants is needed. Rapid advances in sequencing approaches and bioinformatic tools now allow estimation of telomere content from whole-genome sequencing (WGS) data, a proxy for telomere length. While telomere content has been quantified extensively using quantitative polymerase chain reaction (qPCR) and WGS in humans, no study to date has compared the effectiveness of WGS in estimating telomere length in plants relative to qPCR approaches. In this study, we use 100 Populus clones re-sequenced using short-read Illumina sequencing to quantify telomere length comparing three different bioinformatic approaches (Computel, K-seek and TRIP) in addition to qPCR. Overall, telomere length estimates varied across different bioinformatic approaches, but were highly correlated across methods for individual genotypes. A positive correlation was observed between WGS estimates and qPCR, however, Computel estimates exhibited the greatest correlation. Computel incorporates genome coverage into telomere length calculations, suggesting that genome coverage is likely important to telomere length quantification when using WGS data. Overall, telomere estimates from WGS provided greater precision and accuracy of telomere length estimates relative to qPCR. The findings suggest WGS is a promising approach for assessing telomere length and, as the field of telomere ecology evolves, may provide added value to assaying response to biotic and abiotic environments for plants needed to accelerate plant breeding and conservation management.

Los cambios en la longitud de los telómeros se utilizan cada vez más para indicar la respuesta de las especies al estrés ambiental en diversos taxones. A pesar de este amplio uso, pocos estudios han explorado la longitud de los telómeros en las plantas. Por lo tanto, es necesario evaluar nuevos enfoques para medir los telómeros en las plantas. Los rápidos avances en los enfoques de secuenciación y las herramientas bioinformáticas ahora permiten estimar el contenido de los telómeros a partir de datos de secuenciación del genoma completo (WGS), un indicador de la longitud de los telómeros. Si bien el contenido de los telómeros se ha cuantificado ampliamente mediante la reacción en cadena de la polimerasa cuantitativa (qPCR) y WGS en humanos, ningún estudio hasta la fecha ha comparado la efectividad de WGS para estimar la longitud de los telómeros en plantas en relación con los enfoques de qPCR. En este estudio, utilizamos cien clones de álamos (Populus) resecuenciados mediante secuenciación Illumina de lectura corta para cuantificar la longitud de los telómeros comparando tres diferentes enfoques bioinformáticos, Computel, K-seek y TRIP, además de qPCR. En general, las estimaciones de la longitud de los telómeros variaron según los diferentes enfoques bioinformáticos, pero la longitud de los telómeros estuvo altamente correlacionada entre los métodos para genotipos individuales. Se observó una correlación positiva entre las estimaciones de WGS y qPCR; sin embargo, las estimaciones de Computel mostraron la mayor correlación. Computel incorpora la cobertura del genoma en los cálculos de la longitud de los telómeros, lo que sugiere que la cobertura del genoma probablemente es importante para la cuantificación de la longitud de los telómeros cuando se utilizan datos de WGS. En general, las estimaciones de los telómeros de WGS proporcionaron mayor precisión y exactitud de las estimaciones de la longitud de los telómeros en relación con la qPCR. Los hallazgos sugieren que WGS es un enfoque prometedor para evaluar la longitud de los telómeros y, a medida que evoluciona el campo de la ecología de los telómeros, puede proporcionar un valor agregado para analizar la respuesta a ambientes bióticos y abióticos de las plantas necesarias para acelerar los programas de mejoramiento genético y conservación.

Virological evidence of the impact of non-pharmaceutical interventions against COVID-19 in Ecuador, a resource-limited setting.

Ecuador had substantial COVID-19-mortality during 2020 despite early implementation of non-pharmaceutical interventions (NPIs). Resource-limited settings like Ecuador have high proportions of informal labour which entail high human mobility, questioning efficacy of NPIs. We performed a retrospective observational study in Ecuador's national reference laboratory for viral respiratory infections during March 2020-February 2021 using stored respiratory specimens from 1950 patients, corresponding to 2.3% of all samples analysed within the Ecuadorian national surveillance system per week. During 2020, detection of SARS-CoV-2 (Pearson correlation; r = -0.74; p = 0.01) and other respiratory viruses (Pearson correlation; r = -0.68; p = 0.02) by real-time RT-PCR correlated negatively with NPIs stringency. Among respiratory viruses, adenoviruses (Fisher's exact-test; p = 0.026), parainfluenzaviruses (p = 0.04), enteroviruses (p < 0.0001) and metapneumoviruses (p < 0.0001) occurred significantly more frequently during months of absent or non-stringent NPIs (characterized by <55% stringency according to the Oxford stringency index data for Ecuador). Phylogenomic analyses of 632 newly characterized SARS-CoV-2 genomes revealed 100 near-parallel SARS-CoV-2 introductions during early 2020 in the absence of NPIs. NPI stringency correlated negatively with the number of circulating SARS-CoV-2 lineages during 2020 (r = -0.69; p = 0.02). Phylogeographic reconstructions showed differential SARS-CoV-2 dispersion patterns during 2020, with more short-distance transitions potentially associated with recreational activity during non-stringent NPIs. There were also fewer geographic transitions during strict NPIs (n = 450) than during non-stringent or absent NPIs (n = 580). Virological evidence supports that NPIs had an effect on virus spread and distribution in Ecuador, providing a template for future epidemics in resource-limited settings and contributing to a balanced assessment of societal costs entailed by strict NPIs.

First Report of SARS-CoV-2 Lineage B.1.1.7 (Alpha Variant) in Ecuador, January 2021.

On January 5 2021, Ecuadorian COVID-19 genomic surveillance program detected a suspicious case of the B.1.1.7 lineage (alpha variant) of SARS-CoV-2 in Los Rios province, later confirmed by genome sequencing. The patient travelled from the UK by the end of December 2020. By contact tracing, several new cases were detected confirming B.1.1.7 transmission and spreading in Ecuador.

Analytical and clinical evaluation of a heat shock SARS-CoV-2 detection method without RNA extraction for N and E genes RT-qPCR.

BACKGROUND: The COVID-19 pandemic has caused significant supply shortages worldwide for SARS-CoV-2 molecular diagnosis, like RNA extraction kits. OBJECTIVE: The aim of our study was to evaluate the clinical performance and analytical sensitivity of a simple SARS-CoV-2 diagnosis protocol based on heat shock without RNA extraction using both "CDC" (N gene) and "Charite" (E gene) RT-qPCR protocols. RESULTS: 1,036 nasopharyngeal samples, 543 of them SARS-CoV-2 positive, were analyzed. The heat shock method correctly identified 68.8% (232/337) and 89.4% (202/226) of SARS-CoV-2 positive samples for N gene and E gene, respectively. Analytical sensitivity was assessed for heat shock method using the CDC RT-qPCR protocol, obtaining sensitivity values of 98.6%, 93.3% and 84.8% for limit of detection of 100.000, 50.000 and 20.000 viral RNA copies/mL of sample. CONCLUSIONS: Our findings show that a simple heat shock SARS-CoV-2 RT-qPCR diagnosis method without RNA extraction is a reliable alternative for potentially infectious SARS-CoV-2 positive patients at the time of testing. This affordable protocol can help overcome the cost and supply shortages for SARS-CoV-2 diagnosis, especially in developing countries. In Ecuador, it has been used already by laboratories in the public health system for more than 100.000 specimens.

Comparative Plastid Genomics of Neotropical Bulbophyllum (Orchidaceae; Epidendroideae).

Pantropical Bulbophyllum, with ∼2,200 species, is one of the largest genera in Orchidaceae. Although phylogenetics and taxonomy of the ∼60 American species in the genus are generally well understood, some species complexes need more study to clearly delimit their component species and provide information about their evolutionary history. Previous research has suggested that the plastid genome includes phylogenetic markers capable of providing resolution at low taxonomic levels, and thus it could be an effective tool if these divergent regions can be identified. In this study, we sequenced the complete plastid genome of eight Bulbophyllum species, representing five of six Neotropical taxonomic sections. All plastomes conserve the typical quadripartite structure, and, although the general structure of plastid genomes is conserved, differences in ndh-gene composition and total length were detected. Total length was determined by contraction and expansion of the small single-copy region, a result of an independent loss of the seven ndh genes. Selection analyses indicated that protein-coding genes were generally well conserved, but in four genes, we identified 95 putative sites under positive selection. Furthermore, a total of 54 polymorphic simple sequence repeats were identified, for which we developed amplification primers. In addition, we propose 10 regions with potential to improve phylogenetic analyses of Neotropical Bulbophyllum species.

Characterization of sequence variability hotspots in Cranichideae plastomes (Orchidaceae, Orchidoideae).

This study reports complete plastome sequences for six species of Neotropical Cranichideae and focuses on identification of the most variable regions (hotspots) in this group of orchids. These structure of these six plastomes is relatively conserved, exhibiting lengths ranging between 142,599 to 154,562 bp with 36.7% GC on average and exhibiting typical quadripartite arrangement (LSC, SSC and two IRs). Variation detected in the LSC/IR and SSC/IR junctions is explained by the loss of ndhF and ycf1 length variation. For the two genera of epiphytic clade in Spiranthinae, almost whole sets of the ndh-gene family were missing. Eight mutation hotspots were identified based on nucleotide diversity, sequence variability and parsimony-informative sites. Three of them (rps16-trnQ, trnT-trnL, rpl32-trnL) seem to be universal hotspots in the family, and the other five (trnG-trnR, trnR-atpA, trnP-psaJ, rpl32-infA, and rps15-ycf1) are described for the first time as orchid molecular hotspots. These regions have much more variation than all those used previously in phylogenetics of the group and offer useful plastid markers for phylogenetic, barcoding and population genetic studies. The use of whole plastomes or exclusive no-gap matrices also positioned with high support the holomycotrophic Rhizanthella among Orchidoideae plastomes in model-based analyses, showing the utility of plastomes for phylogenetic placement of this unusual genus.