| Téléchargement | - Voir le manuscrit accepté : Next-generation sequencing of 16S ribosomal RNA gene amplicons (PDF, 600 Kio)
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| DOI | Trouver le DOI : https://doi.org/10.3791/51709 |
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| Auteur | Rechercher : Sanschagrin, Sylvie1; Rechercher : Yergeau, Étienne1 |
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| Affiliation | - Conseil national de recherches du Canada. Énergie, les mines et l'environnement
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| Format | Texte, Article |
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| Sujet | Molecular Biology; Issue 90; Metagenomics; Bacteria; 16S ribosomal RNA gene; Amplicon sequencing; Next-generation sequencing; benchtop sequencers |
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| Résumé | One of the major questions in microbial ecology is "who is there?" This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution. |
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| Date de publication | 2014-08-29 |
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| Dans | |
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| Langue | anglais |
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| Publications évaluées par des pairs | Oui |
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| Numéro NPARC | 21272676 |
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| Exporter la notice | Exporter en format RIS |
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| Signaler une correction | Signaler une correction (s'ouvre dans un nouvel onglet) |
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| Identificateur de l’enregistrement | 6a3eed0c-f729-4a8c-800c-cf5765d6e276 |
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| Enregistrement créé | 2014-12-03 |
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| Enregistrement modifié | 2020-06-04 |
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