Phylogenetic relationships of a rabies virus isolate in Costa Rica

Introduction: The sylvatic cycle of rabies is a significant sanitary burden in Central America. The Costa Rican government monitors cases since 1985 and infections from bats are still reported for wild animals, livestock, and humans, generating a need of further pathogen characterization in the region. Objective: To compare rabies phylogenetic analyses from complete genomes with nucleoprotein gene studies. Methods: For the phylogenetic analyses we used four rabies tissue samples collected in 2018, and generated complete genomes by Next-Generation sequencing (NGS). We also extracted RNA from tissues of confirmed cases and generated ssDNA using several primers. Double-stranded DNA was generated and used to generate genomic libraries. Results: We describe, for the first-time, the complete genome of four sequences of the rabies virus isolated in Costa Rica in 2018. Complete genome trees resembled the topology of nucleoprotein gene trees. All isolates were related to Desmodus rotundus. One sample group into Lineage (L)2, and the remaining samples group in L1, matched previous reports from regional rabies viruses. Conclusion: Our method produces valid viral assemblies from clinical specimens without target enrichment or viral isolation.

Novel Nematode-Killing Protein-1 (Nkp-1) from a Marine Epiphytic Bacterium Pseudoalteromonas tunicata

Drug resistance among parasitic nematodes has resulted in an urgent need for the development of new therapies. However, the high re-discovery rate of anti-nematode compounds from terrestrial environments necessitates a new repository for future drug research. Marine epiphytes are hypothesised to produce nematicidal compounds as a defence against bacterivorous predators, thus representing a promising yet underexplored source for anti-nematode drug discovery. The marine epiphytic bacterium Pseudoalteromonas tunicata is known to produce several bioactive compounds. Screening heterologously expressed genomic libraries of P. tunicata against the nematode Caenorhabditis elegans, identified as an E. coli clone (HG8), shows fast-killing activity. Here we show that clone HG8 produces a novel nematode-killing protein-1 (Nkp-1) harbouring a predicted carbohydrate-binding domain with weak homology to known bacterial pore-forming toxins. We found bacteria expressing Nkp-1 were able to colonise the C. elegans intestine, with exposure to both live bacteria and protein extracts resulting in physical damage and necrosis, leading to nematode death within 24 h of exposure. Furthermore, this study revealed C. elegans dar (deformed anal region) and internal hatching may act as a nematode defence strategy against Nkp-1 toxicity. The characterisation of this novel protein and putative mode of action not only contributes to the development of novel anti-nematode applications in the future but reaffirms the potential of marine epiphytic bacteria as a new source of novel biomolecules.

A magnet to draw a bright needle out from the haystack -- RADOrgMiner, an automated pipeline to genotype organellar reads from RADseq data

Different versions of Restriction-site Associated DNA sequencing (RADseq) have become powerful and popular tools in molecular ecology. Although RADseq datasets are regarded as representative of the nuclear genome, reduced representation genomic libraries may also sample the organellar (mitochondrial and, in case of plants, plastid) DNA. Extraction of organellar loci from RADseq data can provide additional insights into the phylogenetics of the study group which comes at no additional sequencing effort. Cytoplasmic genetic variance can help better understand the evolutionary history by uncovering past hybridization and identifying the maternal (or, rarely, the paternal) lineage due to rapid lineage sorting. We developed a pipeline in bash that is based on existing bioinformatic tools to automatically mine and genotype organellar loci contained RADseq libraries. The utility of our pipeline is tested on eight, publicly available datasets spanning different phylogenetic levels (i.e. from family-level phylogenies to phylogeography) and RADseq methods (sdRAD, ddRAD, ezRAD, GBS) for genotyping both mitochondrial and plastid loci, which were subject to phylogenetic tree reconstruction. In all cases, organellar phylogenies adequately supplemented the original studies either by corroborating the large-scale picture based on RADseq or by bringing additional evidence on past or contemporary hybridization. RADseq methods designed to achieve a larger horizontal coverage (i.e. ddRAD, ezRAD) evidently yielded longer organellar alignments, but sdRAD and GBS still provided useful polymorphic loci found in the cytoplasmic DNA. Our newly developed pipeline for the above purpose can be run under a Unix-line operating system and is freely accessible at https://github.com/laczkol/RADOrgMiner

A High-Throughput Skim-sequencing Approach for Genotyping, Dosage Estimation and Identifying Translocations

The development of next generation sequencing (NGS) enabled a shift from array-based genotyping to high-throughput genotyping by directly sequencing genomic libraries. Even though whole genome sequencing was initially too costly for routine analysis in large populations, such as those utilized for breeding or genetic studies, continued advancements in genome sequencing and bioinformatics have provided the opportunity to utilize whole-genome information. As new sequencing platforms can routinely provide high-quality sequencing data for sufficient genome coverage, a limitation comes in the time and high cost of library construction when multiplexing a large number of samples. Here we describe a high-throughput whole-genome skim-sequencing (skim-seq) approach that can be utilized for a broad range of genotyping and genomic characterization. Using optimized low-volume Illumina Nextera chemistry, we developed a skim-seq method and combined up to 960 samples in one multiplex library using dual index barcoding. With the dual-index barcoding, the number of samples for multiplexing can be adjusted depending on amount of data required and extended to 3,072 samples or more. Panels of double haploid wheat lines (Triticum aestivum, CDC Stanley x CDC Landmark), wheat-barley (T. aestivum x Hordeum vulgare) and wheat-wheatgrass (Triticum durum x Thinopyrum intermedium) introgression lines as well as known monosomic wheat stocks were genotyped using the skim-seq approach. Bioinformatics pipelines were developed for various applications where sequencing coverage ranged from 1x down to 0.01x per sample. Using reference genomes, we detected chromosome dosage, identified aneuploidy, and karyotyped introgression lines from the low coverage skim-seq data. Leveraging the recent advancements in genome sequencing, skim-seq provides an effective and low-cost tool for routine genotyping and genetic analysis, which can track and identify introgressions and genomic regions of interest in genetics research and applied breeding programs.

A magnet to draw a bright needle out from the haystack -- RADOrgMiner, an automated pipeline to genotype organellar reads from RADseq data

Different versions of Restriction-site Associated DNA sequencing (RADseq) have become powerful and popular tools in molecular ecology. Although RADseq datasets are regarded as representative of the nuclear genome, reduced representation genomic libraries may also sample the organellar (mitochondrial and, in case of plants, plastid) DNA. Extraction of organellar loci from RADseq data can provide additional insights into the phylogenetics of the study group which comes at no additional sequencing effort. Cytoplasmic genetic variance can help better understand the evolutionary history by uncovering past hybridization and identifying the maternal (or, rarely, the paternal) lineage due to rapid lineage sorting. We developed a pipeline in bash that is based on existing bioinformatic tools to automatically mine and genotype organellar loci contained RADseq libraries. The utility of our pipeline is tested on eight, publicly available datasets spanning different phylogenetic levels (i.e. from family-level phylogenies to phylogeography) and RADseq methods (sdRAD, ddRAD, ezRAD, GBS) for genotyping both mitochondrial and plastid loci, which were subject to phylogenetic tree reconstruction. In all cases, organellar phylogenies adequately supplemented the original studies either by corroborating the large-scale picture based on RADseq or by bringing additional evidence on past or contemporary hybridization. RADseq methods designed to achieve a larger horizontal coverage (i.e. ddRAD, ezRAD) evidently yielded longer organellar alignments, but sdRAD and GBS still provided useful polymorphic loci found in the cytoplasmic DNA. Our newly developed pipeline for the above purpose can be run under a Unix-line operating system and is freely accessible at https://github.com/laczkol/RADOrgMiner

Multiplexing microbial gDNA with SQK-RBK004 rapid kit v1

Purpose This protocol is intended to guide users in creating genomic libraries for MinION & GridION sequencing, using Oxford Nanopore’s SQK-RBK004 rapid kit with high molecular weight microbial gDNA.

Two-step functional screen on multiple proteinaceous substrates reveals temperature-robust proteases with a broad-substrate range

To support the bio-based industry in development of environment-friendly processes and products, an optimal toolbox of biocatalysts is key. Although functional screen of (meta)genomic libraries may potentially contribute to identifying new enzymes, the discovery of new enzymes meeting industry compliance demands is still challenging. This is particularly noticeable in the case of proteases, for which the reports of metagenome-derived proteases with industrial applicability are surprisingly limited. Indeed, proteolytic clones have been typically assessed by its sole activity on casein or skim milk and limited to mild screening conditions. Here, we demonstrate the use of six industry-relevant animal and plant by-products, namely bone, feather, blood meals, gelatin, gluten, and zein, as complementary substrates in functional screens and show the utility of temperature as a screening parameter to potentially discover new broad-substrate range and robust proteases for the biorefinery industry. By targeting 340,000 clones from two libraries of pooled isolates of mesophilic and thermophilic marine bacteria and two libraries of microbial communities inhabiting marine environments, we identified proteases in four of eleven selected clones that showed activity against all substrates herein tested after prolonged incubation at 55 °C. Following sequencing, in silico analysis and recombinant expression in Escherichia coli, one functional protease, 58% identical at sequence level to previously reported homologs, was found to readily hydrolyze highly insoluble zein at temperatures up to 50 °C and pH 9–11. It is derived from a bacterial group whose ability to degrade zein was unknown. This study reports a two-step screen resulting in identification of a new marine metagenome-derived protease with zein-hydrolytic properties at common biomass processing temperatures that could be useful for the modern biorefinery industry. Key points • A two-step multi-substrate strategy for discovery of robust proteases. • Feasible approach for shortening enzyme optimization to industrial demands. • A new temperature-tolerant protease efficiently hydrolyzes insoluble zein.

Comparative Analysis of SNP Discovery and Genotyping in Fagus sylvatica L. and Quercus robur L. Using RADseq, GBS, and ddRAD Methods

Next-generation sequencing of reduced representation genomic libraries (RRL) is capable of providing large numbers of genetic markers for population genetic studies at relatively low costs. However, one major concern of these types of markers is the precision of genotyping, which is related to the common problem of missing data, which appears to be particularly important in association and genomic selection studies. We evaluated three RRL approaches (GBS, RADseq, ddRAD) and different SNP identification methods (de novo or based on a reference genome) to find the best solutions for future population genomics studies in two economically and ecologically important broadleaved tree species, namely F. sylvatica and Q. robur. We found that the use of ddRAD method coupled with SNP calling based on reference genomes provided the largest numbers of markers (28 k and 36 k for beech and oak, respectively), given standard filtering criteria. Using technical replicates of samples, we demonstrated that more than 80% of SNP loci should be considered as reliable markers in GBS and ddRAD, but not in RADseq data. According to the reference genomes’ annotations, more than 30% of the identified ddRAD loci appeared to be related to genes. Our findings provide a solid support for using ddRAD-based SNPs for future population genomics studies in beech and oak.