Microbiota profiling with long amplicons using Nanopore sequencing

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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Authors: Cuscó, Anna|||0000-0002-9574-5755, Catozzi, Carlotta|||0000-0002-1028-1489, Viñes, Joaquim|||0000-0003-0709-5236, Sánchez Bonastre, Armando|||0000-0001-9160-1124, Francino, Olga|||0000-0002-9022-3835
Format: article
Publication Date:2019
Country:España
Institution:Universitat Autònoma de Barcelona
Repository:Dipòsit Digital de Documents de la UAB
Language:English
OAI Identifier:oai:ddd.uab.cat:253255
Online Access:https://ddd.uab.cat/record/253255
https://dx.doi.org/urn:doi:10.12688/f1000research.16817.2
Access Level:Open access
Keyword:Microbiome
Microbiota
16S
Rrn operon
Nanopore
Canine
Low-biomass
Skin
Dog
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spelling Microbiota profiling with long amplicons using Nanopore sequencingfull-length 16S rRNA gene and the 16S-ITS-23S of the rrn operonCuscó, Anna|||0000-0002-9574-5755Catozzi, Carlotta|||0000-0002-1028-1489Viñes, Joaquim|||0000-0003-0709-5236Sánchez Bonastre, Armando|||0000-0001-9160-1124Francino, Olga|||0000-0002-9022-3835MicrobiomeMicrobiota16SRrn operonNanoporeCanineLow-biomassSkinDogBackground: 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. 22019-01-0120192019-01-01Articlehttp://purl.org/coar/resource_type/c_6501VoRhttp://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/articleapplication/pdfhttps://ddd.uab.cat/record/253255https://dx.doi.org/urn:doi:10.12688/f1000research.16817.2reponame:Dipòsit Digital de Documents de la UABinstname:Universitat Autònoma de BarcelonaInglésengAgència de Gestió d'Ajuts Universitaris i de Recerca https://doi.org/10.13039/501100003030 2017DI037Agència de Gestió d'Ajuts Universitaris i de Recerca https://doi.org/10.13039/501100003030 2013DI011open accesshttp://purl.org/coar/access_right/c_abf2Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.https://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:ddd.uab.cat:2532552026-06-06T12:50:31Z
dc.title.none.fl_str_mv Microbiota profiling with long amplicons using Nanopore sequencing
full-length 16S rRNA gene and the 16S-ITS-23S of the rrn operon
title Microbiota profiling with long amplicons using Nanopore sequencing
spellingShingle Microbiota profiling with long amplicons using Nanopore sequencing
Cuscó, Anna|||0000-0002-9574-5755
Microbiome
Microbiota
16S
Rrn operon
Nanopore
Canine
Low-biomass
Skin
Dog
title_short Microbiota profiling with long amplicons using Nanopore sequencing
title_full Microbiota profiling with long amplicons using Nanopore sequencing
title_fullStr Microbiota profiling with long amplicons using Nanopore sequencing
title_full_unstemmed Microbiota profiling with long amplicons using Nanopore sequencing
title_sort Microbiota profiling with long amplicons using Nanopore sequencing
dc.creator.none.fl_str_mv Cuscó, Anna|||0000-0002-9574-5755
Catozzi, Carlotta|||0000-0002-1028-1489
Viñes, Joaquim|||0000-0003-0709-5236
Sánchez Bonastre, Armando|||0000-0001-9160-1124
Francino, Olga|||0000-0002-9022-3835
author Cuscó, Anna|||0000-0002-9574-5755
author_facet Cuscó, Anna|||0000-0002-9574-5755
Catozzi, Carlotta|||0000-0002-1028-1489
Viñes, Joaquim|||0000-0003-0709-5236
Sánchez Bonastre, Armando|||0000-0001-9160-1124
Francino, Olga|||0000-0002-9022-3835
author_role author
author2 Catozzi, Carlotta|||0000-0002-1028-1489
Viñes, Joaquim|||0000-0003-0709-5236
Sánchez Bonastre, Armando|||0000-0001-9160-1124
Francino, Olga|||0000-0002-9022-3835
author2_role author
author
author
author
dc.subject.none.fl_str_mv Microbiome
Microbiota
16S
Rrn operon
Nanopore
Canine
Low-biomass
Skin
Dog
topic Microbiome
Microbiota
16S
Rrn operon
Nanopore
Canine
Low-biomass
Skin
Dog
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.
publishDate 2019
dc.date.none.fl_str_mv 2
2019-01-01
2019
2019-01-01
dc.type.none.fl_str_mv Article
http://purl.org/coar/resource_type/c_6501
VoR
http://purl.org/coar/version/c_970fb48d4fbd8a85
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv https://ddd.uab.cat/record/253255
https://dx.doi.org/urn:doi:10.12688/f1000research.16817.2
url https://ddd.uab.cat/record/253255
https://dx.doi.org/urn:doi:10.12688/f1000research.16817.2
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.relation.none.fl_str_mv Agència de Gestió d'Ajuts Universitaris i de Recerca https://doi.org/10.13039/501100003030 2017DI037
Agència de Gestió d'Ajuts Universitaris i de Recerca https://doi.org/10.13039/501100003030 2013DI011
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
https://creativecommons.org/licenses/by/4.0/
dc.rights.openaire.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv open access
http://purl.org/coar/access_right/c_abf2
https://creativecommons.org/licenses/by/4.0/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.source.none.fl_str_mv reponame:Dipòsit Digital de Documents de la UAB
instname:Universitat Autònoma de Barcelona
instname_str Universitat Autònoma de Barcelona
reponame_str Dipòsit Digital de Documents de la UAB
collection Dipòsit Digital de Documents de la UAB
repository.name.fl_str_mv
repository.mail.fl_str_mv
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