The microbial species described to date represent only a small fraction of Earth's total biodiversity. To expand this knowledge, DNA barcoding is a powerful method for multiple species identification. It is based on the amplification and sequencing of conserved regions. Unfortunately, the conventional short-reads sequencing leads to a limited taxonomic resolution due to fragmentary sequences, but the emergence of new long-reads sequencing techniques, such as Nanopore sequencing, improves this aspect. Indeed, long-reads, by including a bigger part of the standardized marker sequence, enhance the accuracy of species identification and increase the possibility to identify low abundance species. However, compared to earlier technologies, bioinformatic tools specifically designed for Nanopore sequences’ analysis are lacking. Therefore, in our work, we focused on setting an amplicon-Nanopore sequencing workflow and developed a pipeline able to process long-noisy reads. To test and compare our pipeline with the existing ones, the 16S and ITS gene of several known microorganisms, with different taxonomic similarity, was amplified and sequenced using Nanopore technology. The produced sequences were then analyzed with our pipeline as well as available software. The results showed a precision and a recall close to 1 comparable to the Emu software. Then, we tested our method's flexibility and reliability by applying it using other selected genetic markers to achieve deep identification at the pathovar/subspecies level for quarantine bacterial pathogens. Our approach proved promising for the precise identification of microorganisms at the genus-species level for broader metabarcoding studies and at a deeper taxonomic resolution: pathovar or sequence type.
Long-reads Nanopore amplicon-sequencing for a rapid and precise identification of multiple microorganisms / Crosara, V.; Tatulli, G.; Scala, V.; Reverberi, M.; Pucci, N.; Loreti, S.; Faino, L.. - (2024). (Intervento presentato al convegno XXIX Convegno Nazionale SIPaV tenutosi a Trento, Italia).
Long-reads Nanopore amplicon-sequencing for a rapid and precise identification of multiple microorganisms
V. Crosara
;M. Reverberi;L. Faino
2024
Abstract
The microbial species described to date represent only a small fraction of Earth's total biodiversity. To expand this knowledge, DNA barcoding is a powerful method for multiple species identification. It is based on the amplification and sequencing of conserved regions. Unfortunately, the conventional short-reads sequencing leads to a limited taxonomic resolution due to fragmentary sequences, but the emergence of new long-reads sequencing techniques, such as Nanopore sequencing, improves this aspect. Indeed, long-reads, by including a bigger part of the standardized marker sequence, enhance the accuracy of species identification and increase the possibility to identify low abundance species. However, compared to earlier technologies, bioinformatic tools specifically designed for Nanopore sequences’ analysis are lacking. Therefore, in our work, we focused on setting an amplicon-Nanopore sequencing workflow and developed a pipeline able to process long-noisy reads. To test and compare our pipeline with the existing ones, the 16S and ITS gene of several known microorganisms, with different taxonomic similarity, was amplified and sequenced using Nanopore technology. The produced sequences were then analyzed with our pipeline as well as available software. The results showed a precision and a recall close to 1 comparable to the Emu software. Then, we tested our method's flexibility and reliability by applying it using other selected genetic markers to achieve deep identification at the pathovar/subspecies level for quarantine bacterial pathogens. Our approach proved promising for the precise identification of microorganisms at the genus-species level for broader metabarcoding studies and at a deeper taxonomic resolution: pathovar or sequence type.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.