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Abstract: Objective: This study aimed to employ advanced bioinformatics and modern sequencing approaches to solve a diagnostic problem of persistent Campylobacter spp. molecular detection yet negative culture results from four consecutive stool samples of a previously healthy patient with newly diagnosed selective IgA deficiency and prolonged diarrhoea. Methods: Metagenomic next-generation sequencing (mNGS) based on short-paired end reads with basic bioinformatic read classification analysis was used at first. Due to ambiguous results, advanced bioinformatics involving contigs construction and classification, reference genome mappings and reads filtering with BBSplit, additionally coupled with metagenomic long-reads sequencing and Full-length 16S rRNA metabarcoding were employed to further elucidate the results. Virulence factors were analysed using the Prokka Genome Annotation tool. Modified classical bacteriology methods were finally used for further clarification. Results: Short-pair end reads analysis identified several Campylobacter species in all four samples. After advanced bioinformatic approaches were applied, candidatus C. infans was suspected as the putative pathogen. This result was further supported by metagenomic long-reads sequencing and Full-length 16S rRNA metabarcoding. Nevertheless, after modifying the culture conditions based on mNGS results, a mixed culture of candidatus C. infans and C.ureolyticus was obtained. Sequencing of the mixed culture resulted in an 87.48% and 73.47% genome coverage of candidatus C. infans and C. ureolyticus, respectively. In the candidatus C. infans genome more virulence factors hits were found than in the C. ureolyticus genome thus supporting the first as the most probable cause of symptoms. Conclusion: This study shows the pivotal role and strengths of mNGS in unravelling an unusual case of diarrhoea and demonstrates how mNGS can guide established microbiological methods to improve on current limitations. However, it also emphasises the need for careful interpretation of sequencing data, particularly for closely related bacterial species from clinical samples that are known to support complex microbial communities.
Keywords: 16S rRNA metabarcoding, bioinformatics, campylobacter, metagenomics, NGS
Published in DiRROS: 15.04.2026; Views: 196; Downloads: 163
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Abstract: The mucilage phenomenon consists of the appearance of large gelatinous aggregates floating in the water column. In summer 2024, this event has reappeared in the Northern Adriatic Sea (NAS) on a large scale. This study provides an integrated characterization of the microalgal community within mucilage aggregates and surrounding waters in two NAS areas (Gulf of Trieste and Conero Riviera) using light microscopy (LM), metabarcoding (MB) based on ribosomal 18S V4 and V9 markers, and scanning electron microscopy (SEM) for selected taxa identification. Mucilage aggregates acted as dynamic microbial hotspots, hosting a rich diatom community, with abundances 1–2 orders of magnitude higher than in the surrounding water. Dominant diatom species were Cylindrotheca closterium, Nitzschia spp., Nitzschia gobbii, and Thalassionema nitzschioides. Some phytoflagellates (e.g., Tetraselmis spp.) and dinoflagellates (e.g., Karlodinium veneficum, Pseliodinium fusus, and Wangodinium sinense) were detected exclusively by MB, while LM and SEM revealed species missed by other approaches. Gonyaulax fragilis, one of the species indicated as an important mucus producer, was present at the onset and throughout the phenomenon, as detected by LM and MB. The integrated approach improves knowledge of microalgal communities in NAS mucilage.
Keywords: mucilage, Northern Adriatic Sea, phytoplankton, microalgae, metabarcoding, microscopy, Gonyaulax fragilis, Harmful Algal Blooms
Published in DiRROS: 27.01.2026; Views: 539; Downloads: 445
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Abstract: DNA metabarcoding of benthic diatoms has been successfully applied for biomonitoring at the national scale and can now be considered technically ready for routine application. However, protocols and methods still vary between and within countries, limiting their transferability and the comparability of results. In order to overcome this, routine use of DNA metabarcoding for diatom biomonitoring requires knowledge of the sources of variability introduced by the different steps of the procedure. Here, we examine how elements of routine procedures contribute to variability between European laboratories. A set of four experiments were performed focusing on DNA extraction and PCR amplification steps to evaluate their reproducibility between different laboratories and the variability introduced by different protocols currently applied by the scientific community. Under the guidance of a reference laboratory, 17 participants from 14 countries performed DNA extraction and PCR amplification in parallel, using the same fixed protocol and their own choice of protocol. Experiments were performed by each participant on a set of standardised DNA and biofilm samples (river, lake and mock community) to investigate potential systematic and random errors. Our results revealed the successful transferability of a protocol amongst labs and a highly similar and consistent ecological assessment outcome obtained regardless of the protocols used by each participant. We propose an “all for one but prove them all” strategy, suggesting that distinct protocols can be used within the scientific community, as long as their consistency is be proven by following minimum standard requirements.
Keywords: cross-laboratory experiment, DNA-based approach, ecological status assessment, intercalibration, standardisation, environmental science, biomonitoring, DNA metabarcoding, benthic diatoms
Published in DiRROS: 17.01.2025; Views: 953; Downloads: 821
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Abstract: DNA metabarcoding combines DNA barcoding with high-throughput sequencing to identify different taxa within environmental communities. The ITS has already been proposed and widely used as universal barcode marker for plants, but a comprehensive, updated and accurate reference dataset of plant ITS sequences has not been available so far. Here, we constructed reference datasets of Viridiplantae ITS1, ITS2 and entire ITS sequences including both Chlorophyta and Streptophyta. The sequences were retrieved from NCBI, and the ITS region was extracted. The sequences underwent identity check to remove misidentified records and were clustered at 99% identity to reduce redundancy and computational effort. For this step, we developed a script called ‘better clustering for QIIME’ (bc4q) to ensure that the representative sequences are chosen according to the composition of the cluster at a different taxonomic level. The three datasets obtained with the bc4q script are PLANiTS1 (100 224 sequences), PLANiTS2 (96 771 sequences) and PLANiTS (97 550 sequences), and all are pre-formatted for QIIME, being this the most used bioinformatic pipeline for metabarcoding analysis. Being curated and updated reference databases, PLANiTS1, PLANiTS2 and PLANiTS are proposed as a reliable, pivotal first step for a general standardization of plant DNA metabarcoding studies. The bc4q script is presented as a new tool useful in each research dealing with sequences clustering.
Keywords: DNA metabarcoding, reference datasets
Published in DiRROS: 19.07.2024; Views: 2337; Downloads: 2117
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