Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and the 16S-ITS-23S of the rrn operon [version 2; peer review: 2 approved, 3 approved with reservations]
Background: Profiling the microbiome of low-biomass samples is challenging for metagenomics since these samples are prone to contain DNA from other sources (e.g. host or environment). The usual approach is sequencing short regions of the 16S rRNA gene, which fails to assign taxonomy to genus and spe...
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doaj-fe3f4d3e13ad42148e71b05c4d67a9322020-11-25T03:46:59ZengF1000 Research LtdF1000Research2046-14022019-08-01710.12688/f1000research.16817.221950Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and the 16S-ITS-23S of the rrn operon [version 2; peer review: 2 approved, 3 approved with reservations]Anna Cuscó0Carlotta Catozzi1Joaquim Viñes2Armand Sanchez3Olga Francino4Vetgenomics, SL, Bellaterra (Cerdanyola del Vallès), Barcelona, 08193, SpainDipartimento di Medicina Veterinaria, Università degli Studi di Milano, Milano, ItalyVetgenomics, SL, Bellaterra (Cerdanyola del Vallès), Barcelona, 08193, SpainMolecular Genetics Veterinary Service (SVGM), Universitat Autonoma of Barcelona, Bellaterra (Cerdanyola del Vallès), Barcelona, 08193, SpainMolecular Genetics Veterinary Service (SVGM), Universitat Autonoma of Barcelona, Bellaterra (Cerdanyola del Vallès), Barcelona, 08193, SpainBackground: Profiling the microbiome of low-biomass samples is challenging for metagenomics since these samples are prone to contain DNA from other sources (e.g. host or environment). The usual approach is sequencing short regions of the 16S rRNA gene, which fails to assign taxonomy to genus and species level. To achieve an increased taxonomic resolution, we aim to develop long-amplicon PCR-based approaches using Nanopore sequencing. We assessed two different genetic markers: the full-length 16S rRNA (~1,500 bp) and the 16S-ITS-23S region from the rrn operon (4,300 bp). Methods: We sequenced a clinical isolate of Staphylococcus pseudintermedius, two mock communities and two pools of low-biomass samples (dog skin). Nanopore sequencing was performed on MinION™ using the 1D PCR barcoding kit. Sequences were pre-processed, and data were analyzed using EPI2ME or Minimap2 with rrn database. Consensus sequences of the 16S-ITS-23S genetic marker were obtained using canu. Results: The full-length 16S rRNA and the 16S-ITS-23S region of the rrn operon were used to retrieve the microbiota composition of the samples at the genus and species level. For the Staphylococcus pseudintermedius isolate, the amplicons were assigned to the correct bacterial species in ~98% of the cases with the16S-ITS-23S genetic marker, and in ~68%, with the 16S rRNA gene when using EPI2ME. Using mock communities, we found that the full-length 16S rRNA gene represented better the abundances of a microbial community; whereas, 16S-ITS-23S obtained better resolution at the species level. Finally, we characterized low-biomass skin microbiota samples and detected species with an environmental origin. Conclusions: Both full-length 16S rRNA and the 16S-ITS-23S of the rrn operon retrieved the microbiota composition of simple and complex microbial communities, even from the low-biomass samples such as dog skin. For an increased resolution at the species level, targeting the 16S-ITS-23S of the rrn operon would be the best choice.https://f1000research.com/articles/7-1755/v2 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Anna Cuscó Carlotta Catozzi Joaquim Viñes Armand Sanchez Olga Francino |
spellingShingle |
Anna Cuscó Carlotta Catozzi Joaquim Viñes Armand Sanchez Olga Francino Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and the 16S-ITS-23S of the rrn operon [version 2; peer review: 2 approved, 3 approved with reservations] F1000Research |
author_facet |
Anna Cuscó Carlotta Catozzi Joaquim Viñes Armand Sanchez Olga Francino |
author_sort |
Anna Cuscó |
title |
Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and the 16S-ITS-23S of the rrn operon [version 2; peer review: 2 approved, 3 approved with reservations] |
title_short |
Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and the 16S-ITS-23S of the rrn operon [version 2; peer review: 2 approved, 3 approved with reservations] |
title_full |
Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and the 16S-ITS-23S of the rrn operon [version 2; peer review: 2 approved, 3 approved with reservations] |
title_fullStr |
Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and the 16S-ITS-23S of the rrn operon [version 2; peer review: 2 approved, 3 approved with reservations] |
title_full_unstemmed |
Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and the 16S-ITS-23S of the rrn operon [version 2; peer review: 2 approved, 3 approved with reservations] |
title_sort |
microbiota profiling with long amplicons using nanopore sequencing: full-length 16s rrna gene and the 16s-its-23s of the rrn operon [version 2; peer review: 2 approved, 3 approved with reservations] |
publisher |
F1000 Research Ltd |
series |
F1000Research |
issn |
2046-1402 |
publishDate |
2019-08-01 |
description |
Background: Profiling the microbiome of low-biomass samples is challenging for metagenomics since these samples are prone to contain DNA from other sources (e.g. host or environment). The usual approach is sequencing short regions of the 16S rRNA gene, which fails to assign taxonomy to genus and species level. To achieve an increased taxonomic resolution, we aim to develop long-amplicon PCR-based approaches using Nanopore sequencing. We assessed two different genetic markers: the full-length 16S rRNA (~1,500 bp) and the 16S-ITS-23S region from the rrn operon (4,300 bp). Methods: We sequenced a clinical isolate of Staphylococcus pseudintermedius, two mock communities and two pools of low-biomass samples (dog skin). Nanopore sequencing was performed on MinION™ using the 1D PCR barcoding kit. Sequences were pre-processed, and data were analyzed using EPI2ME or Minimap2 with rrn database. Consensus sequences of the 16S-ITS-23S genetic marker were obtained using canu. Results: The full-length 16S rRNA and the 16S-ITS-23S region of the rrn operon were used to retrieve the microbiota composition of the samples at the genus and species level. For the Staphylococcus pseudintermedius isolate, the amplicons were assigned to the correct bacterial species in ~98% of the cases with the16S-ITS-23S genetic marker, and in ~68%, with the 16S rRNA gene when using EPI2ME. Using mock communities, we found that the full-length 16S rRNA gene represented better the abundances of a microbial community; whereas, 16S-ITS-23S obtained better resolution at the species level. Finally, we characterized low-biomass skin microbiota samples and detected species with an environmental origin. Conclusions: Both full-length 16S rRNA and the 16S-ITS-23S of the rrn operon retrieved the microbiota composition of simple and complex microbial communities, even from the low-biomass samples such as dog skin. For an increased resolution at the species level, targeting the 16S-ITS-23S of the rrn operon would be the best choice. |
url |
https://f1000research.com/articles/7-1755/v2 |
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