Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and whole rrn operon [version 1; referees: 2 approved, 1 approved with reservations]
Background: Profiling the microbiome of low-biomass samples is challenging for metagenomics since these samples often contain DNA from other sources, such as the host or the environment. The usual approach is sequencing specific hypervariable regions of the 16S rRNA gene, which fails to assign taxon...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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F1000 Research Ltd
2018-11-01
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| Series: | F1000Research |
| Online Access: | https://f1000research.com/articles/7-1755/v1 |
| _version_ | 1819296401378508800 |
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| author | Anna Cuscó Carlotta Catozzi Joaquim Viñes Armand Sanchez Olga Francino |
| author_facet | Anna Cuscó Carlotta Catozzi Joaquim Viñes Armand Sanchez Olga Francino |
| author_sort | Anna Cuscó |
| collection | DOAJ |
| description | Background: Profiling the microbiome of low-biomass samples is challenging for metagenomics since these samples often contain DNA from other sources, such as the host or the environment. The usual approach is sequencing specific hypervariable regions of the 16S rRNA gene, which fails to assign taxonomy to genus and species level. Here, we aim to assess long-amplicon PCR-based approaches for assigning taxonomy at the genus and species level. We use Nanopore sequencing with two different markers: full-length 16S rRNA (~1,500 bp) and the whole rrn operon (16S rRNA–ITS–23S rRNA; 4,500 bp). Methods: We sequenced a clinical isolate of Staphylococcus pseudintermedius, two mock communities (HM-783D, Bei Resources; D6306, ZymoBIOMICS™) and two pools of low-biomass samples (dog skin from either the chin or dorsal back), using the MinION™ sequencer 1D PCR barcoding kit. Sequences were pre-processed, and data were analyzed using the WIMP workflow on EPI2ME or Minimap2 software with rrn database. Results: The full-length 16S rRNA and the rrn operon were used to retrieve the microbiota composition at the genus and species level from the bacterial isolate, mock communities and complex skin samples. For the Staphylococcus pseudintermedius isolate, when using EPI2ME, the amplicons were assigned to the correct bacterial species in ~98% of the cases with the rrn operon marker, and in ~68% of the cases with the 16S rRNA gene. In both skin microbiota samples, we detected many species with an environmental origin. In chin, we found different Pseudomonas species in high abundance, whereas in dorsal skin there were more taxa with lower abundances. Conclusions: Both full-length 16S rRNA and 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, using the rrn operon would be the best choice. |
| first_indexed | 2024-12-24T04:57:31Z |
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| id | doaj.art-ddf1c689d36a4cc4890dbbd8c3cf78de |
| institution | Directory Open Access Journal |
| issn | 2046-1402 |
| language | English |
| last_indexed | 2024-12-24T04:57:31Z |
| publishDate | 2018-11-01 |
| publisher | F1000 Research Ltd |
| record_format | Article |
| series | F1000Research |
| spelling | doaj.art-ddf1c689d36a4cc4890dbbd8c3cf78de2022-12-21T17:14:22ZengF1000 Research LtdF1000Research2046-14022018-11-01710.12688/f1000research.16817.118384Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and whole rrn operon [version 1; referees: 2 approved, 1 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 often contain DNA from other sources, such as the host or the environment. The usual approach is sequencing specific hypervariable regions of the 16S rRNA gene, which fails to assign taxonomy to genus and species level. Here, we aim to assess long-amplicon PCR-based approaches for assigning taxonomy at the genus and species level. We use Nanopore sequencing with two different markers: full-length 16S rRNA (~1,500 bp) and the whole rrn operon (16S rRNA–ITS–23S rRNA; 4,500 bp). Methods: We sequenced a clinical isolate of Staphylococcus pseudintermedius, two mock communities (HM-783D, Bei Resources; D6306, ZymoBIOMICS™) and two pools of low-biomass samples (dog skin from either the chin or dorsal back), using the MinION™ sequencer 1D PCR barcoding kit. Sequences were pre-processed, and data were analyzed using the WIMP workflow on EPI2ME or Minimap2 software with rrn database. Results: The full-length 16S rRNA and the rrn operon were used to retrieve the microbiota composition at the genus and species level from the bacterial isolate, mock communities and complex skin samples. For the Staphylococcus pseudintermedius isolate, when using EPI2ME, the amplicons were assigned to the correct bacterial species in ~98% of the cases with the rrn operon marker, and in ~68% of the cases with the 16S rRNA gene. In both skin microbiota samples, we detected many species with an environmental origin. In chin, we found different Pseudomonas species in high abundance, whereas in dorsal skin there were more taxa with lower abundances. Conclusions: Both full-length 16S rRNA and 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, using the rrn operon would be the best choice.https://f1000research.com/articles/7-1755/v1 |
| 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 whole rrn operon [version 1; referees: 2 approved, 1 approved with reservations] F1000Research |
| title | Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and whole rrn operon [version 1; referees: 2 approved, 1 approved with reservations] |
| title_full | Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and whole rrn operon [version 1; referees: 2 approved, 1 approved with reservations] |
| title_fullStr | Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and whole rrn operon [version 1; referees: 2 approved, 1 approved with reservations] |
| title_full_unstemmed | Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and whole rrn operon [version 1; referees: 2 approved, 1 approved with reservations] |
| title_short | Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and whole rrn operon [version 1; referees: 2 approved, 1 approved with reservations] |
| title_sort | microbiota profiling with long amplicons using nanopore sequencing full length 16s rrna gene and whole rrn operon version 1 referees 2 approved 1 approved with reservations |
| url | https://f1000research.com/articles/7-1755/v1 |
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