Phylogenetic analysis of variable and conserved genomic regions in severe acute respiratory syndrome coronavirus 2 (COVID-19)

SARS-CoV-2 has rapidly spread around the world. Several mutations have been detected in its genome, but they do not seem to affect the abilities of the virus to spread or infect. We aimed to explore the conserved genomic regions in coronavirus that could contain the key strengths of the virus. SARS-CoV-2 sequence data were retrieved from Genbank from the period of December 2019 to March 2020. Phylogenetic analyses were conducted for 207 sequences using MEGAX compared with the reference sequence (MN908947.3- CHN-Wuhan Dec-2019). The analysis included seven important genomic regions, the ORF1ab gene (21,290 bp), S gene (3,822 bp), Orf3a gene (827 bp), E gene (227 bp), M gene (669 bp), and N gene (1,259 bp), which play critical roles in virus invasion and replication. Furthermore, the variant nucleotides and amino acids were detected by MEGAX and BLAST. Through the phylogenetic analysis and amino acid substitution, the ORF1ab gene showed 11 conserved regions and also several variable sites. The E and M genes were mainly conserved, and all sequences were included in one clade, with one or two amino acid variants. Orf3a and the N gene have four conserved sites distributed along the genes. The S gene has 12 mutations and four main large conserved regionsWe conclude that the favored occurrence of mutations at the ORFab and Orf3a genes during the SARS-CoV epidemic is an important mechanism for virus pathogenesis. The E and M proteins have an almost conserved structure, whereas the S and N genes have many conserved regions, which could serve as possible targets for vaccine design for SARS-CoV.

Phylogenetic analysis of variable and conserved genomic regions in severe acute respiratory syndrome coronavirus 2 (COVID-19)

SARS-CoV-2 has rapidly spread around the world. Several mutations have been detected in its genome, but they do not seem to affect the abilities of the virus to spread or infect. We aimed to explore the conserved genomic regions in coronavirus that could contain the key strengths of the virus. SARS-CoV-2 sequence data were retrieved from Genbank from the period of December 2019 to March 2020. Phylogenetic analyses were conducted for 207 sequences using MEGAX compared with the reference sequence (MN908947.3- CHN-Wuhan Dec-2019). The analysis included seven important genomic regions, the ORF1ab gene (21,290 bp), S gene (3,822 bp), Orf3a gene (827 bp), E gene (227 bp), M gene (669 bp), and N gene (1,259 bp), which play critical roles in virus invasion and replication. Furthermore, the variant nucleotides and amino acids were detected by MEGAX and BLAST. Through the phylogenetic analysis and amino acid substitution, the ORF1ab gene showed 11 conserved regions and also several variable sites. The E and M genes were mainly conserved, and all sequences were included in one clade, with one or two amino acid variants. Orf3a and the N gene have four conserved sites distributed along the genes. The S gene has 12 mutations and four main large conserved regionsWe conclude that the favored occurrence of mutations at the ORFab and Orf3a genes during the SARS-CoV epidemic is an important mechanism for virus pathogenesis. The E and M proteins have an almost conserved structure, whereas the S and N genes have many conserved regions, which could serve as possible targets for vaccine design for SARS-CoV.

Pan-Family Assays for Rapid Viral Screening: Reducing Delays in Public Health Responses During Pandemics

Background COVID-19 has highlighted deficiencies in the testing capacity of many developed countries during the early stages of pandemics. Here we describe a strategy utilizing pan-family viral assays to improve early accessibility of large-scale nucleic acid testing. Methods Coronaviruses and SARS-CoV-2 were used as a case-study for assessing utility of pan-family viral assays during the early stages of a novel pandemic. Specificity of a pan-coronavirus (Pan-CoV) assay for a novel pathogen was assessed using the frequency of common human coronavirus (HCoV) species in key populations. A reported Pan-CoV assay was assessed to determine sensitivity to 60 reference coronaviruses, including SARS-CoV-2. The resilience of the primer target regions of this assay to mutation was assessed in 8893 high-quality SARS-CoV-2 genomes to predict ongoing utility during pandemic progression. Results Due to common HCoV species, a Pan-CoV assay would return false positives for as few as 1% of asymptomatic adults, but up to 30% of immunocompromised patients with respiratory disease. Half of reported Pan-CoV assays identify SARS-CoV-2 and with small adjustments can accommodate diverse variation observed in animal coronaviruses. The target region of one well established Pan-CoV assay is highly resistant to mutation compared to species-specific SARS-CoV-2 RT-PCR assays. Conclusions Despite cross-reactivity with common pathogens, pan-family assays may greatly assist management of emerging pandemics through prioritization of high-resolution testing or isolation measures. Targeting highly conserved genomic regions make pan-family assays robust and resilient to mutation. A strategic stockpile of pan-family assays may improve containment of novel diseases prior to the availability of species-specific assays.

Proteomic characterization of chromosomal common fragile site (CFS)-associated proteins uncovers ATRX as a regulator of CFS stability

Common fragile sites (CFSs) are conserved genomic regions prone to break under conditions of replication stress (RS). Thus, CFSs are hotspots for rearrangements in cancer and contribute to its chromosomal instability. Here, we have performed a global analysis of proteins that recruit to CFSs upon mild RS to identify novel players in CFS stability. To this end, we performed Chromatin Immunoprecipitation (ChIP) of FANCD2, a protein that localizes specifically to CFSs in G2/M, coupled to mass spectrometry to acquire a CFS interactome. Our strategy was validated by the enrichment of many known regulators of CFS maintenance, including Fanconi Anemia, DNA repair and replication proteins. Among the proteins identified with unknown functions at CFSs was the chromatin remodeler ATRX. Here we demonstrate that ATRX forms foci at a fraction of CFSs upon RS, and that ATRX depletion increases the occurrence of chromosomal breaks, a phenotype further exacerbated under mild RS conditions. Accordingly, ATRX depletion increases the number of 53BP1 bodies and micronuclei, overall indicating that ATRX is required for CFS stability. Overall, our study provides the first proteomic characterization of CFSs as a valuable resource for the identification of novel regulators of CFS stability.

Identifying Conserved Genomic Elements and Designing Universal Probe Sets To Enrich Them

Targeted enrichment of conserved genomic regions is a popular method for collecting large amounts of sequence data from non-model taxa for phylogenetic, phylogeographic, and population genetic studies. Yet, few open-source workflows are available to identify conserved genomic elements shared among divergent taxa and to design enrichment baits targeting these regions. These shortcomings limit the application of targeted enrichment methods to many organismal groups. Here, I describe a universal workflow for identifying conserved genomic regions in available genomic data and for designing targeted enrichment baits to collect data from these conserved regions. I demonstrate how this computational approach can be applied to diverse organismal groups by identifying sets of conserved loci and designing enrichment baits targeting thousands of these loci in the understudied arthropod groups Arachnida, Coleoptera, Diptera, Hemiptera, or Lepidoptera. I then use in silico analyses to demonstrate that these conserved loci reconstruct the accepted relationships among genome sequences from the focal arthropod orders, and we perform in vitro validation of the Arachnid probe set as part of a separate manuscript (Starrett et al. Submitted). All of the documentation, design steps, software code, and probe sets developed here are available under an open-source license for restriction-free testing and use by any research group, and although the examples in this manuscript focus on understudied and exceptionally diverse arthropod groups, the software workflow is applicable to all organismal groups having some form of pre-existing genomic information.

High Phylogenetic Utility of an Ultraconserved Element Probe Set Designed for Arachnida

Arachnida is an ancient, diverse, and ecologically important animal group that contains a number of species of interest for medical, agricultural, and engineering applications. Despite this applied importance, many aspects of the arachnid tree of life remain unresolved, hindering comparative approaches to arachnid biology. Biologists have made considerable efforts to resolve the arachnid phylogeny; yet, limited and challenging morphological characters, as well as a dearth of genetic resources, have confounded these attempts. Here, we present a genomic toolkit for arachnids featuring hundreds of conserved DNA regions (ultraconserved elements or UCEs) that allow targeted sequencing of any species in the arachnid tree of life. We used recently developed capture probes designed from conserved genomic regions of available arachnid genomes to enrich a sample of loci from 32 diverse arachnids. Sequence capture returned an average of 487 UCE loci for all species, with a range from 170 to 722. Phylogenetic analysis of these UCEs produced a highly resolved arachnid tree with relationships largely consistent with recent transcriptome-based phylogenies. We also tested the phylogenetic informativeness of UCE probes within the spider, scorpion, and harvestman orders, demonstrating the utility of these markers at shallower taxonomic scales, even down to the level of species differences. This probe set will open the door to phylogenomic and population genomic studies across the arachnid tree of life, enabling systematics, species delimitation, species discovery, and conservation of these diverse arthropods.

Analysis of plastome and chondriome genome types in potato somatic hybrids from Solanum tuberosum × Solanum etuberosum

Cytoplasm types of the potato somatic hybrids from Solanum tuberosum × Solanum etuberosum were analysed using chloroplast (cp) and mitochondrial (mt) organelle genomes-specific markers. Of the 29 markers (15 cpDNA and 14 mtDNA) amplified in the 26 genotypes, 5 cpDNA (H3, NTCP4, NTCP8, NTCP9, and ALC1/ALC3) and 13 mtDNA markers showed polymorphism. The cluster analysis based on the mtDNA markers detected higher diversity compared with the cpDNA markers. Presence of new mtDNA fragments of the markers, namely, T11-2, Nsm1, pumD, Nsm3, and Nsm4, were observed, while monomorphic loci revealed highly conserved genomic regions in the somatic hybrids. The study revealed that the somatic hybrids had diverse cytoplasm types consisting predominantly of T-, W-, and C-, with a few A- and S-type cp genomes; and α-, β-, and γ-type mt genomes. Somatic hybridization has unique potential to widen the cytoplasm types of the cultivated gene pools from wild species through introgression by breeding methods.