Biology subjects

D'Andreano, S.

Publications and source records attributed to D'Andreano, S..

2 recordsLinked to original sources

bioRxiv2020

Rapid and real-time identification of fungi up to the species level with long amplicon Nanopore sequencing from clinical samples.

The availability of long-read technologies, like Oxford Nanopore Technologies, provides the opportunity to sequence longer fragments of the fungal ribosomal operon, up to 6 Kb (18S-ITS1-5.8S-ITS2-28S), and to improve the taxonomy assignment of the communities up to the species level and in real-time. We assess the taxonomy skills of amplicons targeting a 3.5 Kb region (V3 18S-ITS1-5.8S-ITS2-28S D2) and a 6 Kb region (V1 18S-ITS1-5.8S-ITS2-28S D12) with the Whats in my pot (WIMP) classifier. We used the ZymoBIOMICS mock community and different microbiological fungal cultures as positive controls. Long amplicon sequencing correctly identified Saccharomyces cerevisiae and Cryptococcus neoformans from the mock community and Malassezia pachydermatis, Microsporum canis, and Aspergillus fumigatus from the microbiological cultures. Besides, we identified Rhodotorula graminis in a culture mislabeled as Candida spp. We applied the same approach to external otitis in dogs. Malassezia was the dominant fungal genus in dogs ear skin, whereas M. pachydermatis was the main species in the healthy sample. Conversely, we identified a higher representation of M. globosa and M. sympodialis in otitis affected samples. We demonstrate the suitability of long ribosomal amplicons to characterize the fungal community of complex samples, either healthy or with clinical signs of infection.

microbiology↗

bioRxiv2017

Using MinION to characterize dog skin microbiota through full-length 16S rRNA gene sequencing approach

The most common strategy to assess microbiota is sequencing specific hypervariable regions of 16S rRNA gene using 2nd generation platforms (such as MiSeq or Ion Torrent PGM). Despite obtaining high-quality reads, many sequences fail to be classified at the genus or species levels due to their short length. This pitfall can be overcome sequencing the full-length 16S rRNA gene (1,500bp) by 3rd generation sequencers.\n\nWe aimed to assess the performance of nanopore sequencing using MinION on characterizing microbiota complex samples. First set-up step was performed using a staggered mock community (HM-783D). Then, we sequenced a pool of several dog skin microbiota samples previously sequenced by Ion Torrent PGM. Sequences obtained for full-length 16S rRNA with degenerated primers retrieved increased richness estimates at high taxonomic level (Bacteria and Archaea) that were missed with short-reads. Besides, we were able to obtain taxonomic assignments down to species level, although it was not always feasible due to: i) incomplete database; ii) primer set chosen; iii) low taxonomic resolution of 16S rRNA gene within some genera; and/or iv) sequencing errors. Nanopore sequencing of the full-length 16S rRNA gene using MinION with 1D sequencing kit allowed us inferring microbiota composition of a complex microbial community to lower taxonomic levels than short-reads from 2nd generation sequencers.

microbiology↗