Validation of Oxford Nanopore MinION technology for a fast and low-cost profiling of mucosal sea bream (Sparus aurata) microbiota
| Autor: | Toxqui-Rodríguez, S., Naya-Català, Fernando, Sitjà-Bobadilla, Ariadna, Piazzon de Haro, María Carla, Pérez-Sánchez, Jaume |
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| Přispěvatelé: | European Commission, Ministerio de Economía y Competitividad (España) |
| Rok vydání: | 2022 |
| Popis: | Resumen del trabajo presentado en 6th International Symposium on Genomics in Aquaculture, celebrado en Granada (España) del 04 al 06 de mayo de 2022. With the growth of the aquaculture industry, there has been a growing interest in manipulating fish gut microbiota to improve welfare and nutrition since it is critical for many host functions such as digestion, nutrient metabolism, disease resistance, and immune function. Accurate and rapid identification of microbial communities through sequencing of the 16S ribosomal RNA (16S rRNA) gene is an approach widely used since the introduction of high-throughput sequencing technologies (HTS). Nowadays, several affordable methods involving different sequencing technologies exist, being 16S amplicon Illumina sequencing the most widely used. However, the choice of sequencing platform and the downstream analysis are known to yield somewhat different results. Of particular interest, Oxford Nanopore MinION Technology offers a low price, portability, and fast sequencing throughput compared to the Illumina MiSeq and PacBio sequencers. Nevertheless, the error rate of the generated amplicon data is higher, and differences in the experimental conditions for 16S rRNA-based PCR could bias microbiota assessments in the samples. To find a suitable solution that could offer reliable results considering the time, cost, and sequencing throughput, this study aimed to 1) Evaluate the feasibility of MinION sequencing by comparing16S amplicon data of gilthead sea bream intestinal microbiota using the Illumina MiSeq, PacBio (performed with standard procedures by sequencing companies), and MinION (on-site sequencing) platforms; and 2) Standardize the experimental 16S protocol for sample preparation and PCR conditions when sequencing microbiota samples with the Oxford Nanopore MinION Technology. Different 16S gene regions were amplified depending on the sequencing platform (V3-V4 for Illumina MiSeq and full 16S rRNA gene for MinION and PacBio), and bioinformatics pipelines were conducted according to each sequencer amplicon data. Concerning the MinION protocol, five PCR conditions were tested using the Nanopore 16S barcoding kit, combining different temperatures, cycling conditions, and nesting. For all samples, the taxonomic assignment was made using the SILVA (http://www.arb-silva.de) database v. 138. The coming results will provide precise insights into the advantages and disadvantages of this specific sequencing platform, and their suitability depending on the experimental approach, expected output, and cost and time constraints of a given experiment. EATFISH (H2020 #956697), AQUAEXCEL3.0 (H2020 #871108), RYC2018-024049-I & ESF. |
| Databáze: | OpenAIRE |
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