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.

Microalgae are currently considered one of the most promising resources for biofuel production, aquaculture feedstock and new pharmaceuticals. Among them, green algae of the genus Tetraselmis are extensively studied for their lipid accumulation in nutrient-starvation conditions. In this paper, we present the full-transcriptome of Tetraselmis suecica and differential expression analysis between nitrogen-starved and -repleted conditions (at stationary phase) focusing not only on lipid metabolism but giving new insights on nutrient starvation responses. Transcripts involved in signal transduction pathways, stress and antioxidant responses and solute transport were strongly up-regulated when T . suecica was cultured under nitrogen starvation. On the contrary, transcripts involved in amino acid synthesis, degradation of sugars, secondary metabolite synthesis, as well as photosynthetic activity were down-regulated under the same conditions. Among differentially expressed transcripts, a polyketide synthase and three lipoxygenases (involved in the synthesis of secondary metabolites with antipredator, anticancer and anti-infective activities) were identified, suggesting the potential synthesis of bioactive compounds by this microalga. In addition, the transcript for a putative nitrilase, enzyme used in nitrile bioremediation, is here reported for the first time for T. suecica . These findings give new insights on T. suecica responses to nutrient starvation and on possible biotechnological applications for green algae.

Neuroblastoma (NB) is an aggressive infancy tumor, leading cause of death among preschool age diseases. Here we focused on characterization of exosomal DNA (exo-DNA) isolated from plasma cell-derived exosomes of neuroblastoma patients, and its potential use for detection of somatic mutations present in the parental tumor cells. Exosomes are small extracellular membrane vesicles secreted by most cells, playing an important role in intercellular communications. Using an enzymatic method, we provided evidence for the presence of double-stranded DNA in the NB exosomes. Moreover, by whole exome sequencing, we demonstrated that NB exo-DNA represents the entire exome and that it carries tumor-specific genetic mutations, including those occurring on known oncogenes and tumor suppressor genes in neuroblastoma (ALK, CHD5, SHANK2, PHOX2B, TERT, FGFR1, and BRAF). NB exo-DNA can be useful to identify variants responsible for acquired resistance, such as mutations of ALK, TP53, and RAS/MAPK genes that appear in relapsed patients. The possibility to isolate and to enrich NB derived exosomes from plasma using surface markers, and the quick and easy extraction of exo-DNA, gives this methodology a translational potential in the clinic. Exo-DNA can be an attractive non-invasive biomarker for NB molecular diagnostic, especially when tissue biopsy cannot be easily available.

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.

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.