Bioinformatics approaches for hybrid de novo genome assembly

De novo genome assembly, the computational process to reconstruct the genomic sequence from scratch stitching together overlapping reads, plays a key role in computational biology and, to date, it cannot be considered a solved problem. Many bioinformatics approaches are available to deal with different type of data generated by diverse technologies. Assemblies relying on short read data resulted to be highly fragmented, reconstructing short contigs interrupted in repetitive region; on the other side long-read based approaches still suffer of high sequencing error rate, worsening the final consensus quality. This thesis aimed to assess the impact of different assembly approaches on the reconstruction of a highly repetitive genome, identifying the strengths and limiting the weaknesses of such approaches through the integration of orthogonal data types. Moreover, a benchmarking study has been undertaken to improve the contiguity of this genome, describing the improvements obtained thanks to the integration of additional data layers. Assemblies performed using short reads confirmed the limitation in the reconstruction of long sequences for both the software adopted. The use of long reads allowed to improve the genome assembly contiguity, reconstructing also a greater number of gene models. Despite the enhancement of contiguity, base level accuracy of long reads-based assembly could still not reach higher levels. Therefore, short reads were integrated within the assembly process to limit the base level errors present in the reconstructed sequences up to 96%. To order and orient the assembled polished contigs into longer scaffolds, data derived from three different technologies (linked read, chromosome conformation capture and optical mapping) have been analysed. The best contiguity metrics were obtained using chromosome conformation data, which permit to obtain chromosome-scale scaffolds. To evaluate the obtained results, data derived from linked reads and optical mapping have been used to identify putative misassemblies in the scaffolds. Both the datasets allowed the identification of misassemblies, highlighting the importance of integrating data derived from orthogonal technologies in the de novo assembly process. 4 This work underlines the importance of adopting bioinformatics approaches able to deal with data type generated by different technologies. In this way, results could be more accurately validated for the reconstruction of assemblies that could be eventually considered reference genomes

Real-Time On-Site Diagnosis of Quarantine Pathogens in Plant Tissues by Nanopore-Based Sequencing

Rapid and sensitive assays for the identification of plant pathogens are necessary for the effective management of crop diseases. The main limitation of current diagnostic testing is the inability to combine broad and sensitive pathogen detection with the identification of key strains, pathovars, and subspecies. Such discrimination is necessary for quarantine pathogens, whose management is strictly dependent on genotype identification. To address these needs, we have established and evaluated a novel all-in-one diagnostic assay based on nanopore sequencing for the detection and simultaneous characterization of quarantine pathogens, using Xylella fastidiosa as a case study. The assay proved to be at least as sensitive as standard diagnostic tests and the quantitative results agreed closely with qPCR-based analysis. The same sequencing results also allowed discrimination between subspecies when present either individually or in combination. Pathogen detection and typing were achieved within 13 min of sequencing owing to the use of an internal control that allowed to stop sequencing when sufficient data had accumulated. These advantages, combined with the use of portable equipment, will facilitate the development of next-generation diagnostic assays for the efficient monitoring of other plant pathogens

A Rapid and Accurate MinION-Based Workflow for Tracking Species Biodiversity in the Field

Genetic markers (DNA barcodes) are often used to support and confirm species identification. Barcode sequences can be generated in the field using portable systems based on the Oxford Nanopore Technologies (ONT) MinION sequencer. However, to achieve a broader application, current proof-of-principle workflows for on-site barcoding analysis must be standardized to ensure a reliable and robust performance under suboptimal field conditions without increasing costs. Here, we demonstrate the implementation of a new on-site workflow for DNA extraction, PCR-based barcoding, and the generation of consensus sequences. The portable laboratory features inexpensive instruments that can be carried as hand luggage and uses standard molecular biology protocols and reagents that tolerate adverse environmental conditions. Barcodes are sequenced using MinION technology and analyzed with ONTrack, an original de novo assembly pipeline that requires as few as 1000 reads per sample. ONTrack-derived consensus barcodes have a high accuracy, ranging from 99.8 to 100%, despite the presence of homopolymer runs. The ONTrack pipeline has a user-friendly interface and returns consensus sequences in minutes. The remarkable accuracy and low computational demand of the ONTrack pipeline, together with the inexpensive equipment and simple protocols, make the proposed workflow particularly suitable for tracking species under field conditions

USE OF COLLABORATIVE ROBOTS IN INDUSTRY 4.0 : literature analysis and planning of a case study for quality control

openRobotica collaborativa,Analisi della letteratura,Caso studioCollaborative robotics, Literature analysis, Case stud

A Long-Read Sequencing Approach for Direct Haplotype Phasing in Clinical Settings

The reconstruction of individual haplotypes can facilitate the interpretation of disease risks; however, high costs and technical challenges still hinder their assessment in clinical settings. Second-generation sequencing is the gold standard for variant discovery but, due to the production of short reads covering small genomic regions, allows only indirect haplotyping based on statistical methods. In contrast, third-generation methods such as the nanopore sequencing platform developed by Oxford Nanopore Technologies (ONT) generate long reads that can be used for direct haplotyping, with fewer drawbacks. However, robust standards for variant phasing in ONT-based target resequencing efforts are not yet available. In this study, we presented a streamlined proof-of-concept workflow for variant calling and phasing based on ONT data in a clinically relevant 12-kb region of the APOE locus, a hotspot for variants and haplotypes associated with aging-related diseases and longevity. Starting with sequencing data from simple amplicons of the target locus, we demonstrated that ONT data allow for reliable single-nucleotide variant (SNV) calling and phasing from as little as 60 reads, although the recognition of indels is less efficient. Even so, we identified the best combination of ONT read sets (600) and software (BWA/Minimap2 and HapCUT2) that enables full haplotype reconstruction when both SNVs and indels have been identified previously using a highly-accurate sequencing platform. In conclusion, we established a rapid and inexpensive workflow for variant phasing based on ONT long reads. This allowed for the analysis of multiple samples in parallel and can easily be implemented in routine clinical practice, including diagnostic testing

Chlorella vulgaris genome assembly and annotation reveals the molecular basis for metabolic acclimation to high light conditions

International audienc

Hybrid genome assembly and annotation of <i>Paenibacillus pasadenensis</i> strain R16 reveals insights on endophytic life style and antifungal activity

<div><p>Bacteria of the <i>Paenibacillus</i> genus are becoming important in many fields of science, including agriculture, for their positive effects on the health of plants. However, there are little information available on this genus compared to other bacteria (such as <i>Bacillus</i> or <i>Pseudomonas</i>), especially when considering genomic information. Sequencing the genomes of plant-beneficial bacteria is a crucial step to identify the genetic elements underlying the adaptation to life inside a plant host and, in particular, which of these features determine the differences between a helpful microorganism and a pathogenic one. In this study, we have characterized the genome of <i>Paenibacillus pasadenensis</i>, strain R16, recently investigated for its antifungal activities and plant-associated features. An hybrid assembly approach was used integrating the very precise reads obtained by Illumina technology and long fragments acquired with Oxford Nanopore Technology (ONT) sequencing. <i>De novo</i> genome assembly based solely on Illumina reads generated a relatively fragmented assembly of 5.72 Mbp in 99 ungapped sequences with an N50 length of 544 Kbp; hybrid assembly, integrating Illumina and ONT reads, improved the assembly quality, generating a genome of 5.75 Mbp, organized in 6 contigs with an N50 length of 3.4 Mbp. Annotation of the latter genome identified 4987 coding sequences, of which 1610 are hypothetical proteins. Enrichment analysis identified pathways of particular interest for the endophyte biology, including the chitin-utilization pathway and the incomplete siderophore pathway which hints at siderophore parasitism. In addition the analysis led to the identification of genes for the production of terpenes, as for example farnesol, that was hypothesized as the main antifungal molecule produced by the strain. The functional analysis on the genome confirmed several plant-associated, plant-growth promotion, and biocontrol traits of strain R16, thus adding insights in the genetic bases of these complex features, and of the <i>Paenibacillus</i> genus in general.</p></div

Transport genes.

<p>Genes related to transport identified in the genome of <i>Paenibacillus pasadenensis</i> strain R16. In normal text are reported the main categories and, when appropriate, the subcategories are indicated in italic.</p

Genome annotation.

<p>Genome annotation.</p

Sporulation genes.

<p>Sporulation genes.</p