Genome sequence of Mesorhizobium mediterraneum strain R31, a nitrogen-fixing rhizobium used as an inoculant for chickpea in Argentina

Here, we report the complete genome sequence of Mesorhizobium mediterraneum R31, a rhizobial strain recommended and used as a commercial inoculant for chickpea in Argentina. The genome consists of 7.25 Mb, distributed into four circular replicons: a chromosome of 6.72 Mbp and three plasmids of 0.29, 0.17, and 0.07 Mbp.Instituto de Microbiología y Zoología Agrícola (IMYZA)Fil: Foresto, Emiliano. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Instituto de Biotecnología Ambiental y Salud. Departamento de Biología Molecular; ArgentinaFil: Revale, Santiago. University of Oxford. Wellcome Centre for Human Genetics; Reino UnidoFil: Nievas, Fiorela. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Instituto de Biotecnología Ambiental y Salud. Departamento de Biología Molecular; ArgentinaFil: Carezzano, María Evangelina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Instituto de Biotecnología Ambiental y Salud. Departamento de Biología Molecular; ArgentinaFil: Puente, Mariana Laura. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola. Laboratorio de Bacterias Promotoras del Crecimiento Vegetal; ArgentinaFil: Alzari, Pedro. Université de Paris. Institut Pasteur. Unité de Microbiologie Structurale; FranciaFil: Martínez, Mariano. Université de Paris. Institut Pasteur. Unité de Microbiologie Structurale; FranciaFil: Ben-Assaya, Mathilde. Université de Paris. Institut Pasteur. Unité de Microbiologie Structurale; FranciaFil: Mornico, Damien. Institut Pasteur. Département Biologie Computationnelle. Hub de Bioinformatique et Biostatistique; FranciaFil: Santoro, Maricel. Max Planck for Chemical Ecology. Department of Biochemistry; FranciaFil: Martínez-Abarca, Francisco. CSIC. Estación Experimental Del Zaidín. Grupo de Ecología Genética de la Rizósfera; EspañaFil: Giordano, Walter. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Instituto de Biotecnología Ambiental y Salud (INBIAS-CONICET). Departamento de Biología Molecular; ArgentinaFil: Bogino, Pablo. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Instituto de Biotecnología Ambiental y Salud (INBIAS-CONICET). Departamento de Biología Molecular; Argentin

Field-Adapted Full Genome Sequencing of Peste-Des-Petits-Ruminants Virus Using Nanopore Sequencing

Peste-des-petits-ruminants virus (PPRV) is currently the focus of a control and eradication program. Full genome sequencing has the opportunity to become a powerful tool in the eradication program by improving molecular epidemiology and the study of viral evolution. PPRV is prevalent in many resource-constrained areas, with long distances to laboratory facilities, which can lack the correct equipment for high-throughput sequencing. Here we present a protocol for near full or full genome sequencing of PPRV. The use of a portable miniPCR and MinION brings the laboratory to the field and in addition makes the production of a full genome possible within 24 h of sampling. The protocol has been successfully used on virus isolates from cell cultures and field isolates from tissue samples of naturally infected goats

Development of novel bioinformatic pipelines for MinION-based DNA barcoding

DNA-barcoding is the process of taxonomic identification based on the sequence of a marker gene. When complex samples are analysed, we refer in particular to meta-barcoding. Barcoding has traditionally been performed with Sanger sequencing platform. The emergence of second-generation sequencing platforms, mainly represented by Illumina, enabled the high-throughput sequencing of hundreds of samples, and allowed the characterization of complex samples through meta-barcoding experiments. However, fragments sequenced with the Illumina platform are shorter than 600 bp, and this greatly limits taxonomic resolution of closely related species. Moreover, both these platforms suffer of long turnaround time, since they require shipping the samples to a sequencing facility, and complex regulations may hamper the export of material out of the country of origin. More recently, Oxford Nanopore Technologies provided the MinION, a portable and cheap third-generation sequencer, which has the potential of overcoming issues of currently available platforms, thanks to the production of long sequencing reads. However, MinION reads suffer of high error rate, therefore suitable analysis pipelines are needed to overcome this issue. In this thesis I describe the development of bioinformatic pipelines for MinION-based DNA barcoding. Starting from the analysis of single samples, I show how improvements both in sequencing chemistry and in software now allow obtaining consensus sequences directly in the field, with accuracy comparable with Sanger. Conversely, when analysing complex samples, sequencing reads cannot be collapsed for reducing the error rate. However, bioinformatic approaches exploiting increased read length largely compensate the higher error rate, resulting in high correlation between MinION and Illumina up to genus level, and a more marked sensitivity of MinION platform to detect spiked-in indicator species. In conclusion, the results presented in this thesis show that bioinformatic pipelines for the analysis of MinION reads can largely mitigate platform issues, paving the way for this platform to become the gold-standard for barcoding in the near future

Benchmarking of human Y-chromosomal haplogroup classifiers with whole-genome and whole-exome sequence data

In anthropological, medical, and forensic studies, the nonrecombinant region of the human Y chromosome (NRY) enables accurate reconstruction of pedigree relationships and retrieval of ancestral information. Using high-throughput sequencing (HTS) data, we present a benchmarking analysis of command-line tools for NRY haplogroup classification. The evaluation was performed using paired Illumina data from whole-genome sequencing (WGS) and whole-exome sequencing (WES) experiments from 50 unrelated donors. Additionally, as a validation, we also used paired WGS/WES datasets of 54 individuals from the 1000 Genomes Project. Finally, we evaluated the tools on data from third-generation HTS obtained from a subset of donors and one reference sample. Our results show that WES, despite typically offering less genealogical resolution than WGS, is an effective method for determining the NRY haplogroup. Y-LineageTracker and Yleaf showed the highest accuracy for WGS data, classifying precisely 98% and 96% of the samples, respectively. Yleaf outperforms all benchmarked tools in the WES data, classifying approximately 90% of the samples. Yleaf, Y-LineageTracker, and pathPhynder can correctly classify most samples (88%) sequenced with third-generation HTS. As a result, Yleaf provides the best performance for applications that use WGS and WES. Overall, our study offers researchers with a guide that allows them to select the most appropriate tool to analyze the NRY region using both second- and third-generation HTS data

Direct RNA Nanopore Sequencing of SARS-CoV-2 Extracted from Critical Material from Swabs

In consideration of the increasing prevalence of COVID-19 cases in several countries and the resulting demand for unbiased sequencing approaches, we performed a direct RNA sequencing (direct RNA seq.) experiment using critical oropharyngeal swab samples collected from Italian pa-tients infected with SARS-CoV-2 from the Palermo region in Sicily. Here, we identified the sequences SARS-CoV-2 directly in RNA extracted from critical samples using the Oxford Nanopore MinION technology without prior cDNA retrotranscription. Using an appropriate bioinformatics pipeline, we could identify mutations in the nucleocapsid (N) gene, which have been reported previously in studies conducted in other countries. In conclusion, to the best of our knowledge, the technique used in this study has not been used for SARS-CoV-2 detection previously owing to the difficulties in the extraction of RNA of sufficient quantity and quality from routine oropharyngeal swabs. Despite these limitations, this approach provides the advantages of true native RNA sequencing and does not include amplification steps that could introduce systematic errors. This study can provide novel information relevant to the current strategies adopted in SARS-CoV-2 next-generation sequencing

Advancing animal tuberculosis surveillance using culture-independent long-read whole-genome sequencing

Acknowledgments Some of the figures (Figures 4–6 and Supplementary Material S1) were generated using BioRender and draw.io, respectively. Funding The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was funded by the Wellcome Foundation (grant #222941/Z/21/Z), the South African Medical Research Council, American Association of Zoo Veterinarians Wild Animal Health Fund [S005651 and S007355], the National Research Foundation South African Research Chair Initiative [grant #86949], and MHM was supported by Wellcome Trust (grant #216634/Z/19/Z). AGL is supported by the EDCTP TESA III network (CSA2020NoE-3104).Peer reviewedPublisher PD

Scalable and versatile container-based pipelines for de novo genome assembly and bacterial annotation. [version 1; peer review: 2 approved, 1 approved with reservations]

Background: Advancements in DNA sequencing technology have transformed the field of bacterial genomics, allowing for faster and more cost effective chromosome level assemblies compared to a decade ago. However, transforming raw reads into a complete genome model is a significant computational challenge due to the varying quality and quantity of data obtained from different sequencing instruments, as well as intrinsic characteristics of the genome and desired analyses. To address this issue, we have developed a set of container-based pipelines using Nextflow, offering both common workflows for inexperienced users and high levels of customization for experienced ones. Their processing strategies are adaptable based on the sequencing data type, and their modularity enables the incorporation of new components to address the community’s evolving needs. Methods: These pipelines consist of three parts: quality control, de novo genome assembly, and bacterial genome annotation. In particular, the genome annotation pipeline provides a comprehensive overview of the genome, including standard gene prediction and functional inference, as well as predictions relevant to clinical applications such as virulence and resistance gene annotation, secondary metabolite detection, prophage and plasmid prediction, and more. Results: The annotation results are presented in reports, genome browsers, and a web-based application that enables users to explore and interact with the genome annotation results. Conclusions: Overall, our user-friendly pipelines offer a seamless integration of computational tools to facilitate routine bacterial genomics research. The effectiveness of these is illustrated by examining the sequencing data of a clinical sample of Klebsiella pneumoniae

Genetic diversification of persistent Mycobacterium abscessus within cystic fibrosis patients

Mycobacterium (M.) abscessus infections in Cystic Fibrosis (CF) patients cause a deterioration of lung function. Treatment of these multidrug-resistant pathogens is associated with severe side-effects, while frequently unsuccessful. Insight on M. abscessus genomic evolvement during chronic lung infection would be beneficial for improving treatment strategies. A longitudinal study enrolling 42 CF patients was performed at a CF center in Berlin, Germany, to elaborate phylogeny and genomic diversification of in-patient M. abscessus. Eleven of the 42 CF patients were infected with M. abscessus. Five of these 11 patients were infected with global human-transmissible M. abscessus cluster strains. Phylogenetic analysis of 88 genomes from isolates of the 11 patients excluded occurrence of M. abscessus transmission among members of the study group. Genome sequencing and variant analysis of 30 isolates from 11 serial respiratory samples collected over 4.5 years from a chronically infected patient demonstrated accumulation of gene mutations. In total, 53 genes exhibiting non-synonymous variations were identified. Enrichment analysis emphasized genes involved in synthesis of glycopeptidolipids, genes from the embABC (arabinosyltransferase) operon, betA (glucose-methanol-choline oxidoreductase) and choD (cholesterol oxidase). Genetic diversity evolved in a variety of virulence- and resistance-associated genes. The strategy of M. abscessus populations in chronic lung infection is not clonal expansion of dominant variants, but to sustain simultaneously a wide range of genetic variants facilitating adaptation of the population to changing living conditions in the lung. Genomic diversification during chronic infection requires increased attention when new control strategies against M. abscessus infections are explored.Peer Reviewe