Benchmarking of five NGS mapping tools for the reference alignment of bacterial outer membrane vesicles-associated small RNAs.

Advances in small RNAs (sRNAs)-related studies have posed a challenge for NGS-related bioinformatics, especially regarding the correct mapping of sRNAs. Depending on the algorithms and scoring matrices on which they are based, aligners are influenced by the characteristics of the dataset and the reference genome. These influences have been studied mainly in eukaryotes and to some extent in prokaryotes. However, in bacteria, the selection of aligners depending on sRNA-seq data associated with outer membrane vesicles (OMVs) and the features of the corresponding bacterial reference genome has not yet been investigated. We selected five aligners: BBmap, Bowtie2, BWA, Minimap2 and Segemehl, known for their generally good performance, to test them in mapping OMV-associated sRNAs from Aliivibrio fischeri to the bacterial reference genome. Significant differences in the performance of the five aligners were observed, resulting in differential recognition of OMV-associated sRNA biotypes in A. fischeri. Our results suggest that aligner(s) should not be arbitrarily selected for this task, which is often done, as this can be detrimental to the biological interpretation of NGS analysis results. Since each aligner has specific advantages and disadvantages, these need to be considered depending on the characteristics of the input OMV sRNAs dataset and the corresponding bacterial reference genome to improve the detection of existing, biologically important OMV sRNAs. Until we learn more about these dependencies, we recommend using at least two, preferably three, aligners that have good metrics for the given dataset/bacterial reference genome. The overlapping results should be considered trustworthy, yet their differences should not be dismissed lightly, but treated carefully in order not to overlook any biologically important OMV sRNA. This can be achieved by applying the intersect-then-combine approach. For the mapping of OMV-associated sRNAs of A. fischeri to the reference genome organized into two circular chromosomes and one circular plasmid, containing copies of sequences with rRNA- and tRNA-related features and no copies of sequences with protein-encoding features, if the aligners are used with their default parameters, we advise avoiding Segemehl, and recommend using the intersect-then-combine approach with BBmap, BWA and Minimap2 to improve the potential for discovery of biologically important OMV-associated sRNAs.

Contrasting Metabolisms in Green and White Leaf Sectors of Variegated Pelargonium zonale-An Integrative Transcriptomic and Metabolomic Study.

The photosynthetically active green leaf (GL) and non-active white leaf (WL) tissues of variegated Pelargonium zonale provide an excellent model system for studying processes associated with photosynthesis and sink-source interactions, enabling the same microenvironmental conditions. By combining differential transcriptomics and metabolomics, we identified the main differences between these two metabolically contrasting tissues. Genes related to photosynthesis and associated pigments, the Calvin-Benson cycle, fermentation, and glycolysis were strongly repressed in WL. On the other hand, genes related to nitrogen and protein metabolism, defence, cytoskeletal components (motor proteins), cell division, DNA replication, repair and recombination, chromatin remodelling, and histone modifications were upregulated in WL. A content of soluble sugars, TCA intermediates, ascorbate, and hydroxybenzoic acids was lower, while the concentration of free amino acids (AAs), hydroxycinnamic acids, and several quercetin and kaempferol glycosides was higher in WL than in GL. Therefore, WL presents a carbon sink and depends on photosynthetic and energy-generating processes in GL. Furthermore, the upregulated nitrogen metabolism in WL compensates for the insufficient energy from carbon metabolism by providing alternative respiratory substrates. At the same time, WL serves as nitrogen storage. Overall, our study provides a new genetic data resource for the use of this excellent model system and for ornamental pelargonium breeding and contributes to uncovering molecular mechanisms underlying variegation and its adaptive ecological value.

Development of multiplex PCR based NGS protocol for whole genome sequencing of West Nile virus lineage 2 directly from biological samples using Oxford Nanopore platform.

West Nile virus (WNV) can affect humans, birds, horses and another mammals, causing asymptomatic infection, mild febrile disease, neurological and systematic disease and death. In order to gain insight into the prevalence of WNV, a monitoring program has been established in the Republic of Serbia. Whole genome sequencing is essential for the molecular epizootiological analysis of virus entry and transmission routes, especially in high-risk regions. This paper describes the development of a multiplex PCR based NGS protocol for whole genome sequencing of WNV lineage 2 directly from biological samples using Oxford Nanopore (ONT) platform. The results obtained using this platform, confirmed by Sanger sequencing, indicate that this protocol can be applied to obtain whole sequences of the WNV genome, even when the virus concentration in the sample is medium, Ct value is approximately 30. The use of this protocol does not require prior virus isolation on cell culture nor the depletion of host nucleic acids.

Identification and profiling of conserved microRNAs in different developmental stages of crown imperial (Fritillaria imperialis L.) using high-throughput sequencing.

BACKGROUND: Novel strategies for improvement of ornamental plants and their properties relay on miRNA control of differential plant gene expression modulation. Still, in response to the same abiotic stresses, some conserved miRNA families show different expression patterns in different plant species. In parallel, the use of deep sequencing technologies reveals new levels of complexity of regulatory networks in plants through identification of new miRNAs. METHODS AND RESULTS: Fritillaria imperialis plants were collected from their natural habitats in Koohrang, Chaharmahal va Bakhtiari, Iran. Several tissues including stamen, pistil, petal, sepal, leaf, stem, bulb and fruit were collected during three developmental stages (stem elongation, flower development and seed head stages). Using RNAseq and qRT-PCR approach, this research revealed 21 conserved miRNAs, matching 15 miRNA families, in Fritilaria imperialis. CONCLUSIONS: The expression of seven conserved miRNAs (Fim-miR156b, Fim-miR159, Fim-miR166a-5p, Fim-miR169d-5p, Fim-miR171c, Fim-miR393 and Fim-miR396e-3p) was further investigated in different tissues and three developmental stages, suggesting different roles for these miRNAs during growth and development of crown imperial. Gained knowledge from this research can open the door to find efficient ways to secure crown imperial survival, preservation and utilization and if proven useful may be applied in other plant species as well.