Variety Of Sequences Research Articles

Accurate prediction of functional effect of single amino acid variants with deep learning

The assessment of functional effect of amino acid variants is a critical biological problem in proteomics for clinical medicine and protein engineering. Although natively occurring variants offer insights into deleterious variants, high-throughput deep mutational experiments enable comprehensive investigation of amino acid variants for a given protein. However, these mutational experiments are too expensive to dissect millions of variants on thousands of proteins. Thus, computational approaches have been proposed, but they heavily rely on hand-crafted evolutionary conservation, limiting their accuracy. Recent advancement in transformers provides a promising solution to precisely estimate the functional effects of protein variants on high-throughput experimental data. Here, we introduce a novel deep learning model, namely Rep2Mut-V2, which leverages learned representation from transformer models. Rep2Mut-V2 significantly enhances the prediction accuracy for 27 types of measurements of functional effects of protein variants. In the evaluation of 38 protein datasets with 118,933 single amino acid variants, Rep2Mut-V2 achieved an average Spearman’s correlation coefficient of 0.7. This surpasses the performance of six state-of-the-art methods, including the recently released methods ESM, DeepSequence and EVE. Even with limited training data, Rep2Mut-V2 outperforms ESM and DeepSequence, showing its potential to extend high-throughput experimental analysis for more protein variants to reduce experimental cost. In conclusion, Rep2Mut-V2 provides accurate predictions of the functional effects of single amino acid variants of protein coding sequences. This tool can significantly aid in the interpretation of variants in human disease studies.

Difficulties Of Enterobacteriaceae Genome Annotation In Deciphering Gastrointestinal Microbiome Datasets Obtained By 16S RRNA Gene Amplicon Sequencing

Sequencing of the 16S rRNA gene amplicon is the cornerstone of the method for studying diverse bacteria in complex microbial communities. However, its use is complicated by an error rate of 10–17% when annotating 16S rRNA gene sequences. In our study, we examined the degree of accuracy of the taxonomic database of Enterobacteriaceae, compiled using the SILVA 132 reference database and a previously obtained dataset, viz. the microbiome of the gastrointestinal tract in adolescents with normal body weight and obesity. Material and Methods — In this study, previously obtained 16S rRNA gene amplicon sequencing data were used, and the deciphering was carried out using the QIIME2 2019.4 platform. Phylogenetic analysis was performed using MEGA X software. Results — Phylogenetic analysis of this family based on the studied V3–V4 fragment was hampered by polyphyly among some genera, and for half of the variants of the amplicon sequences it was not possible to clarify their genus. Statistical analysis did not reveal significant differences between the samples. Conclusion — Although the average values of bacterial genera in the studied groups intuitively differed from each other, statistical analysis did not reveal significant differences between the samples. However, it can be assumed that a more detailed study of taxonomic diversity, taking into account factors, such as enterotype, duration of breastfeeding and family history, may reveal differences in the frequency distribution.

Unsupervised outlier detection applied to SARS-CoV-2 nucleotide sequences can identify sequences of common variants and other variants of interest

As of June 2022, the GISAID database contains more than 11 million SARS-CoV-2 genomes, including several thousand nucleotide sequences for the most common variants such as delta or omicron. These SARS-CoV-2 strains have been collected from patients around the world since the beginning of the pandemic. We start by assessing the similarity of all pairs of nucleotide sequences using the Jaccard index and principal component analysis. As shown previously in the literature, an unsupervised cluster analysis applied to the SARS-CoV-2 genomes results in clusters of sequences according to certain characteristics such as their strain or their clade. Importantly, we observe that nucleotide sequences of common variants are often outliers in clusters of sequences stemming from variants identified earlier on during the pandemic. Motivated by this finding, we are interested in applying outlier detection to nucleotide sequences. We demonstrate that nucleotide sequences of common variants (such as alpha, delta, or omicron) can be identified solely based on a statistical outlier criterion. We argue that outlier detection might be a useful surveillance tool to identify emerging variants in real time as the pandemic progresses.

Development of nanoluciferase-based sensing system that can specifically detect 1α,25-dihydroxyvitamin D in living cells

Previously, we reported a FLucN-LXXLL+LBD-FLucC system that detects VDR ligands using split firefly luciferase techniques, ligand binding domain (LBD) of VDR, and LXXLL sequences that interact with LBD after VDR ligand binding. In vivo, 25-hydroxyvitamin D3 (25(OH)D3) and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) act as VDR ligands that bind to VDR, and regulate bone-related gene expression. Therefore, the amount of 25(OH)D3 and 1α,25(OH)2D3 are indicators of bone-related diseases such as rickets and osteoporosis. In this study, we have developed a novel LgBiT-LXXLL+LBD-SmBiT system using NanoLuc Binary Technology (NanoBiT), which has an emission intensity several times higher than that of the split-type firefly luciferase. Furthermore, by using genetic engineering techniques, we attempted to construct a novel system that can specifically detect 1α,25(OH)2D3. Because histidine residues at positions 305 and 397 play important roles in forming a hydrogen bond with a hydroxyl group at position C25 of 25(OH)D3 and 1α,25(OH)2D3, His305 and His397 were each substituted by other amino acids. Consequently, the three mutant VDRs, H305D, H397N, and H397E were equally useful to detect 1α,25(OH)2D3 specifically. In addition, among the 58 variants of the LXXLL sequences, LPYEGSLLLKLLRAPVEE showed the greatest increase in luminescence upon the addition of 25(OH)D3 or 1α,25(OH)2D3. Thus, our novel system using NanoBiT appear to be useful for detecting native vitamin D or its derivatives.

Relative infectivity of the SARS-CoV-2 Omicron variant in human alveolar cells.

With the continuous emergence of highly transmissible SARS-CoV-2 variants, the comparison of their infectivity has become a critical issue for public health. However, a direct assessment of the viral characteristic has been challenging because of the lack of appropriate experimental models and efficient methods. Here, we integrated human alveolar organoids and single-cell transcriptome sequencing to facilitate the evaluation. In a proof-of-concept study with four highly transmissible SARS-CoV-2 variants, including GR (B.1.1.119), Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (BA.1), a rapid evaluation of the relative infectivity was possible. Our system demonstrates that the Omicron variant is 5- to 7-fold more infectious to human alveolar cells than the other SARS-CoV-2 variants at the initial stage of infection. To our knowledge, for the first time, this study measures the relative infectivity of the Omicron variant under multiple virus co-infection and provides new experimental procedures that can be applied to monitor emerging viral variants.

An Innovative PTD-IVT-mRNA Delivery Platform for CAR Immunotherapy of ErbB(+) Solid Tumor Neoplastic Cells.

Chimeric antigen receptor (CAR) immunotherapy includes the genetic modification of immune cells to carry such a receptor and, thus, recognize cancer cell surface antigens. Viral transfection is currently the preferred method, but it carries the risk of off-target mutagenicity. Other transfection platforms have thus been proposed, such the in vitro transcribed (IVT)-mRNAs. In this study, we exploited our innovative, patented delivery platform to produce protein transduction domain (PTD)-IVT-mRNAs for the expression of CAR on NK-92 cells. CAR T1E-engineered NK-92 cells, harboring the sequence of T1E single-chain fragment variant (scFv) to recognize the ErbB receptor, bearing either CD28 or 4-1BB as co-stimulatory signaling domains, were prepared and assessed for their effectiveness in two different ErbB(+) cancer cell lines. Our results showed that the PTD-IVT-mRNA of CAR was safely transduced and expressed into NK-92 cells. CAR T1E-engineered NK-92 cells provoked high levels of cell death (25-33%) as effector cells against both HSC-3 (oral squamous carcinoma) and MCF-7 (breast metastatic adenocarcinoma) human cells in the co-incubation assays. In conclusion, the application of our novel PTD-IVT-mRNA delivery platform to NK-92 cells gave promising results towards future CAR immunotherapy approaches.

Identification of TFPI as a receptor reveals recombination-driven receptor switching in Clostridioides difficile toxin B variants

Toxin B (TcdB) is a major exotoxin responsible for diseases associated with Clostridioides difficile infection. Its sequence variations among clinical isolates may contribute to the difficulty in developing effective therapeutics. Here, we investigate receptor-binding specificity of major TcdB subtypes (TcdB1 to TcdB12). We find that representative members of subtypes 2, 4, 7, 10, 11, and 12 do not recognize the established host receptor, frizzled proteins (FZDs). Using a genome-wide CRISPR-Cas9-mediated screen, we identify tissue factor pathway inhibitor (TFPI) as a host receptor for TcdB4. TFPI is recognized by a region in TcdB4 that is homologous to the FZD-binding site in TcdB1. Analysis of 206 TcdB variant sequences reveals a set of six residues within this receptor-binding site that defines a TFPI binding-associated haplotype (designated B4/B7) that is present in all TcdB4 members, a subset of TcdB7, and one member of TcdB2. Intragenic micro-recombination (IR) events have occurred around this receptor-binding region in TcdB7 and TcdB2 members, resulting in either TFPI- or FZD-binding capabilities. Introduction of B4/B7-haplotype residues into TcdB1 enables dual recognition of TFPI and FZDs. Finally, TcdB10 also recognizes TFPI, although it does not belong to the B4/B7 haplotype, and shows species selectivity: it recognizes TFPI of chicken and to a lesser degree mouse, but not human, dog, or cattle versions. These findings identify TFPI as a TcdB receptor and reveal IR-driven changes on receptor-specificity among TcdB variants.

Population-based screening in a municipality after a primary school outbreak of the SARS-CoV-2 Alpha variant, the Netherlands, December 2020-February 2021.

An outbreak of SARS-CoV-2 Alpha variant (Pango lineage B.1.1.7) was detected at a primary school (School X) in Lansingerland, the Netherlands, in December 2020. The outbreak was studied retrospectively, and population-based screening was used to assess the extent of virus circulation and decelerate transmission. Cases were SARS-CoV-2 laboratory confirmed and were residents of Lansingerland (November 16th 2020 until February 22th 2021), or had an epidemiological link with School X or neighbouring schools. The SARS-CoV-2 variant was determined using variant PCR or whole genome sequencing. A questionnaire primarily assessed clinical symptoms. A total of 77 Alpha variant cases were found with an epidemiological link to School X, 16 Alpha variant cases linked to the neighbouring schools, and 146 Alpha variant cases among residents of Lansingerland without a link to the schools. The mean number of self-reported symptoms was not significantly different among Alpha variant infected individuals compared to non-Alpha infected individuals. The secondary attack rate (SAR) among Alpha variant exposed individuals in households was 52% higher compared to non-Alpha variant exposed individuals (p = 0.010), with the mean household age, and mean number of children and adults per household as confounders. Sequence analysis of 60 Alpha variant sequences obtained from cases confirmed virus transmission between School X and neighbouring schools, and showed that multiple introductions of the Alpha variant had already taken place in Lansingerland at the time of the study. The alpha variant caused a large outbreak at both locations of School X, and subsequently spread to neighbouring schools, and households. Population-based screening (together with other public health measures) nearly stopped transmission of the outbreak strain, but did not prevent variant replacement in the Lansingerland municipality.

Detection of Apple Hammerhead Viroid, Apple Luteovirus 1 and Citrus Concave Gum-Associated Virus in Apple Propagation Materials and Orchards in the Czech Republic and Hungary.

Grafting cultivars onto rootstocks is a widely used practice by the apple industry predominantly aimed at faster fruit bearing. Using high-throughput sequencing, we revealed the presence of recently described viral agents, namely apple hammerhead viroid (AHVd), apple luteovirus 1 (ALV-1), and citrus concave gum-associated virus (CCGaV), in germplasm collections and production orchards in the Czech Republic and Hungary. The HTS results were validated with RT-(q)PCR, and Northern blotting techniques. To obtain further insight about the presence of these agents, RT-PCR based surveys were carried out and showed their widespread presence alone or in mixed infections. The pathogens were present both in production areas and in feral samples. In addition, rootstock-to-scion transmission of ALV-1 and CCGaV was confirmed using commercial rootstock materials. Phylogenetic relationships based on partial sequences of distinct variants were also investigated. Furthermore, the rosy apple aphid was found to be ALV-1-positive, suggesting that it might be a potential vector of the virus.

Genome Sequence of Bovine Coronavirus Variants from the Nasal Virome of Irish Beef Suckler and Pre-weaned Dairy Calves Clinically Diagnosed with Bovine Respiratory Disease.

We report 24 bovine coronavirus (BCoV) genome sequences from Ireland. BCoV was sequenced directly from nasal swabs that had been collected during a bovine respiratory disease (BRD) outbreak among recently purchased beef suckler and pre-weaned dairy calves.

Pan-Tissue and -Cancer Analysis of ROR1 and ROR2 Transcript Variants Identify Novel Functional Significance for an Alternative Splice Variant of ROR1.

ROR1/2 are putative druggable targets increasing in significance in translational oncology. Expression of ROR1/2 mRNA and transcript variants has not been systematically examined thus far. ROR1/2 transcript variant sequences, signal peptides for cell surface localisation, and mRNA and transcript variant expression were examined in 34 transcriptomic datasets including 33 cancer types and 54 non-diseased human tissues. ROR1/2 have four and eight transcript variants, respectively. ROR1/2 mRNA and transcript variant expression was detected in various non-diseased tissues. Our analysis identifies predominant expression of ROR1 transcript variant ENST00000545203, which lacks a signal peptide for cell surface localisation, rather than the predicted principal variant ENST00000371079. ENST00000375708 is the predominantly expressed transcript variant of ROR2. ROR1/2 expression in healthy human tissues should be carefully considered for safety assessment of targeted therapy. Studies exploring the function and significance of the predominantly expressed ROR1 transcript variant ENST00000545203 are warranted.

Characterization of Leishmania spp. Causing Cutaneous Lesions with a Negative Parasitological Diagnosis in Panama.

A total of 123 DNA samples from Panamanian patients with cutaneous leishmaniasis (CL) lesions were evaluated. These samples were previously confirmed with CL by a specific KDNA-Viannia PCR but had a negative parasitological diagnosis (Group A). Epidemiological variables, such as age, sex, geographic origin, evolution time, and the number and location of the lesions, were analyzed. No significant differences (p < 0.05) were found when these variables were evaluated against a control panel of 123 CL lesion samples from CL patients with positive parasitological diagnoses (Group B). Of the 123 samples (Group A), 67% (82/123) gave positive results when re-analyzed by PCR-hsp70. An analysis of 69 of these samples via PCR-hsp70-RFLP showed that 59.4% (41/69) of the found restriction patterns corresponded to Leishmania (Viannia) panamensis and 40.6% (28/69) to Leishmania (Viannia) guyanensis. Finally, the sequence and phylogenetic analysis of 32 of the samples confirmed the species in 21 (65.6%, 21/32) samples, originally characterized as L. (V.) panamensis. However, 11 samples (34.4%, 11/32), initially identified via RFLP-Hsp70 as L. (V.) guyanensis, matched the sequence of a genetic variant known as Leishmania sp.1. These results point out the species/genetic variants of Leishmania in the case of CL lesions with an apparently low parasite load.

Phylodynamic analysis of SARS-CoV-2 spread in Rio de Janeiro, Brazil, highlights how metropolitan areas act as dispersal hubs for new variants.

During the first semester of 2021, all of Brazil has suffered an intense wave of COVID-19 associated with the Gamma variant. In July, the first cases of Delta variant were detected in the state of Rio de Janeiro. In this work, we have employed phylodynamic methods to analyse more than 1 600 genomic sequences of Delta variant collected until September in Rio de Janeiro to reconstruct how this variant has surpassed Gamma and dispersed throughout the state. After the introduction of Delta, it has initially spread mostly in the homonymous city of Rio de Janeiro, the most populous of the state. In a second stage, dispersal occurred to mid- and long-range cities, which acted as new close-range hubs for spread. We observed that the substitution of Gamma by Delta was possibly caused by its higher viral load, a proxy for transmissibility. This variant turnover prompted a new surge in cases, but with lower lethality than was observed during the peak caused by Gamma. We reason that high vaccination rates in the state of Rio de Janeiro were possibly what prevented a higher number of deaths.

Optimization and Identification of Single Mutation in Hemoglobin Variants with 2,2,2 Trifluoroethanol Modified Digestion Method and Nano-LC Coupled MALDI MS/MS.

Background: Hemoglobin (Hb) variants arise due to point mutations in globin chains and their pathological treatments rely heavily on the identification of the nature and location of the mutation in the globin chains. Traditional methods for diagnosis such as HPLC and electrophoresis have their own limitations. Therefore, the present study aims to develop and optimize a specific method of sample processing that could lead to improved sequence coverage and analysis of Hb variants by nano LC−MALDI MS/MS. Methods: In our study, we primarily standardized various sample processing methods such as conventional digestion with trypsin followed by 10% acetonitrile treatment, digestion with multiple proteases like trypsin, Glu−C, Lys−C, and trypsin digestion subsequent to 2,2,2 trifluoroethanol (TFE) treatment. Finally, the peptides were identified by LC−MALDI MS/MS. All of these sample processing steps were primarily tested with recombinant Hb samples. After initial optimization, we found that the TFE method was the most suitable one and the efficiency of this method was applied in Hb variant identification based on high sequence coverage. Results: We developed and optimized a method using an organic solvent TFE and heat denaturation prior to digestion, resulting in 100% sequence coverage in the β−chains and 95% sequence coverage in the α−chains, which further helped in the identification of Hb mutations. A Hb variant protein sequence database was created to specify the search and reduce the search time. Conclusion: All of the mutations were identified using a bottom−up non−target approach. Therefore, a sensitive, robust and reproducible method was developed to identify single substitution mutations in the Hb variants from the sequence of the entire globin chains. Biological Significance: Over 330,000 infants are born annually with hemoglobinopathies and it is the major cause of morbidity and mortality in early childhood. Hb variants generally arise due to point mutation in the globin chains. There is high sequence homology between normal Hb and Hb variant chains. Due to this high homology between the two forms, identification of variants by mass spectrometry is very difficult and requires the full sequence coverage of α− and β−chains. As such, there is a need for a suitable method that provides 100% sequence coverage of globin chains for variant analysis by mass spectrometry. Our study provides a simple, robust, and reproducible method that is suitable for LC−MALDI and provides nearly complete sequence coverage in the globin chains. This method may be used in the near future in routine diagnosis for Hb variant analysis.

Variant-Specific Analysis Reveals a Novel Long-Range RNA-RNA Interaction in SARS-CoV-2 Orf1a.

Since the start of the COVID-19 pandemic, understanding the pathology of the SARS-CoV-2 RNA virus and its life cycle has been the priority of many researchers. Currently, new variants of the virus have emerged with various levels of pathogenicity and abundance within the human-host population. Although much of viral pathogenicity is attributed to the viral Spike protein’s binding affinity to human lung cells’ ACE2 receptor, comprehensive knowledge on the distinctive features of viral variants that might affect their life cycle and pathogenicity is yet to be attained. Recent in vivo studies into the RNA structure of the SARS-CoV-2 genome have revealed certain long-range RNA-RNA interactions. Using in silico predictions and a large population of SARS-CoV-2 sequences, we observed variant-specific evolutionary changes for certain long-range RRIs. We also found statistical evidence for the existence of one of the thermodynamic-based RRI predictions, namely Comp1, in the Beta variant sequences. A similar test that disregarded sequence variant information did not, however, lead to significant results. When performing population-based analyses, aggregate tests may fail to identify novel interactions due to variant-specific changes. Variant-specific analyses can result in de novo RRI identification.

Variation in Ovine DGAT1 and Its Association with Carcass Muscle Traits in Southdown Sheep.

Diacylglycerol O-acyltransferase 1 (DGAT1) is a microsomal enzyme that plays a key role in the synthesis of triglycerides. Its gene (DGAT1) is regarded as a candidate gene for variation in milk and meat traits in cattle. The objective of this study was to use a PCR single-strand conformation polymorphism approach to explore sequence variation in two regions of ovine DGAT1 and to assess its effect on meat traits in New Zealand Southdown sheep. Three variant nucleotide sequences were identified in each region, with two single nucleotide polymorphisms (SNPs) and one nucleotide deletion being detected in intron 1 and two SNPs being found in exon 17. The effect of the exon 17 variation was not investigated due to one variant being predominant and the other two variants occurring at low frequencies. In intron 1, one variant (B1) was found to be associated with increase loin meat yield, suggesting that this may have value as a gene marker for improving meat traits.

The glutamate metabotropic receptor 5 (GRM5) gene is associated with beef cattle home range and movement tortuosity

BackgroundThe grazing behaviour of herbivores and their grazing personalities might in part be determined genetically, but there are few studies in beef cattle illustrating this. In this study, we investigated for first time the genetic variation within a candidate ‘grazing gene’, the glutamate metabotropic receptor 5 gene (GRM5), and tested associations between variation in that gene and variation in grazing personality behaviours (GP-behaviours) displayed by free-ranging cows during winter grazing in the steep and rugged rangelands of New Zealand. Mature beef cows (n = 303, from 3 to 10 years of age) were tracked with global positioning system (GPS) and, with 5-minutes (min) relocation frequency, various GP-behaviours were calculated. These included horizontal and vertical distances travelled, mean elevation, elevation range, elevation gain, slope, home range and movement tortuosity, variously calculated using daily relocation trajectories with repeated measurements (i.e., 7 to 24 days (d)) and satellite-derived digital elevation models (DEM). The different GP-behaviours were fitted into mixed models to ascertain their associations with variant sequences and genotypes of GRM5.ResultsWe discovered three GRM5 variants (A, B and C) and identified the six possible genotypes in the cattle studied. The mixed models revealed that A was significantly associated with elevation range, home range and movement tortuosity. Similarly, GRM5 genotypes were associated (P < 0.05) to home range and movement tortuosity, while trends suggesting association (P < 0.1) were also revealed for elevation range and horizontal distance travelled. Most GP-behaviour models were improved by correcting for cow age-class as a fixed factor. The analysis of GP-behaviours averaged per cow age-class suggests that grazing personality is fully established as beef cows reached 4 years of age. Home range and movement tortuosity were not only associated with GRM5 variation, but also negatively correlated with each other (r = − 0.27, P < 0.001).ConclusionsThere seems to be a genetically determined trade-off between home range and movement tortuosity that may be useful in beef cattle breeding programmes aiming to improve the grazing distribution and utilisation of steep and rugged rangelands.

Structural topological analysis of spike proteins of SARS-CoV-2 variants of concern highlight distinctive amino acid substitution patterns

Since the onset of pandemic in 2019, SARS-CoV-2 has diverged into numerous variants driven by antigenic and infectivity-oriented selection. Some variants have accumulated fitness-enhancing mutations, evaded immunity and spread despite global vaccination campaigns. The spike (S) glycoprotein of SARS-CoV-2 demonstrated the greatest immunogenicity and amino acid substitution diversity owing to its importance in the interaction with human angiotensin receptor 2 (hACE2). The S protein consistently emerges as an amino acid substitution (AAS) hotspot in all six lineages, however, in Omicron this enrichment is significantly higher. This study attempts to design and validate a method of mapping S-protein substitution profile across variants to identify the conserved and AAS regions. A substitution matrix was created based on publicly available databases, and the substitution localization was illustrated on a cryo-electron microscopy generated S-protein model. Our analyses indicated that the diversity of N-terminal (NTD) and receptor-binding (RBD) domains exceeded that of any other regions but still contained extended low substitution density regions particularly considering significantly broader substitution profiles of Omicron BA.2 and BA.4/5. Finally, the substitution matrix was compared to a random sample alignment of variant sequences, revealing discrepancies. Therefore, it was suggested to improve matrix accuracy by processing a large number of S-protein sequences using an automated algorithm. Several critical immunogenic and receptor-interacting residues were identified in the conserved regions within NTD and RBD. In conclusion, the structural and topological analysis of S proteins of SARS-CoV-2 variants highlight distinctive amino acid substitution patterns which may be foundational in predicting future variants.

Accurate Cultivar Authentication of Jujube Fruits Using Nano-Fluidic Genotyping of Single Nucleotide Polymorphism (SNP) Markers

Chinese jujube (Ziziphus jujuba Mill.) is an economically important fruit tree that is highly adapted to marginal crop lands and can be grown under a very broad range of climate conditions. Jujube fruits produced from several traditional cultivars in Ningxia, China have enjoyed a premium market price due to their unique flavor and quality attributes. One problem with the high-end jujube market is the adulteration of premium cultivars. The accurate identification of the genetic identity of single jujube fruits is essential for cultivar traceability and authentication. The multi-locus SNP barcoding approach offers an effective solution for cultivar authentication of jujube fruits. To identify variant SNP sequences a nanofluidic array approach was used to test the efficacy of this method with DNA extracted from the fruit pulp of eight jujube cultivars from Ningxia, China. The SNP marker profiles identified the genetic identity of each fruit unambiguously based on multilocus matching and ordination analysis. Results for repeated sampling of multiple fruits from the same tree (using independent DNA extractions) showed a high level of concordance, which demonstrated the reliability of SNP based genotyping platform. This method can handle 100 s to 1000 s of samples per day (based on the platform used). It is robust and cost-effective and has a high potential for its practical application in the jujube industry. The developed method and protocol can be readily applied for botanical authentication of other stone fruits in general.

Phylogeography and genomic analysis of SARS-CoV-2 delta variant in Morocco.

Since the COVID-19 pandemic began in December 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continuously evolved with many variants of concern emerging across the world. In order to monitor the evolution of these variants in Morocco, we analyzed a total of 2130 genomes of the delta variant circulating around the world. We also included 164 Moroccan delta variant sequences in our analysis. Our findings suggest at least four introductions from multiple international sources and a rise of a dominant delta sub-lineage AY.33 in Morocco. Moreover, we report three mutations in the N-terminal domain of the S protein specific to the Moroccan AY.33 isolates, T29A, T250I and T299I. The effect of these mutations on the secondary structure and the dynamic behavior of the S protein N-terminal domain was further determined. We conclude that these mutations might have functional consequences on the S protein of SARS-CoV-2.