Using MinION<sup>™</sup>to characterize dog skin microbiota through full-length 16S rRNA gene sequencing approach

Cuscó, Anna; Viñes, Joaquim; D’Andreano, Sara; Riva, Federica; Casellas, J.; Sánchez, Armand; Francino, Olga

doi:10.1101/167015

Cited by 20 publications

(19 citation statements)

References 63 publications

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“…Actinomyces odontolyticus and Rhodobacter sphaeroides seem to not amplify properly, neither with 16S rRNA gene or rrn operon. Previous studies also detected the same pattern for these specific bacteria even when using or comparing different primer sets [16, 21]. Overall, 16S rRNA primer set seemed less biased than rrn operon.…”

Section: Discussionsupporting

confidence: 68%

“…Several studies targeting the full-length 16S rRNA gene have already been performed using nanopore sequencing to: i) characterize artificial and already characterized bacterial communities (mock community) [15–17]; ii) characterize complex microbiota samples, from mouse gut [18], wastewater [19], microalgae [20] and dog skin [21]; and iii) characterize the pathogenic agent in a clinical sample [22–24]. On the other hand, only two studies have been performed using the whole rrn operon to characterize mock communities [25] and complex natural communities [26].…”

Section: Introductionmentioning

confidence: 99%

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Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and whole rrn operon

Cuscó¹,

Catozzi

Viñes

et al. 2018

Preprint

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Background. Profiling microbiome on low biomass samples is challenging for metagenomics since these samples are prone to present 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 longamplicon 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 gene -ITS -23S rRNA gene; 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 lowbiomass samples (dog skin). Nanopore sequencing was performed on MinION™ (Oxford Nanopore Technologies) using 1D PCR barcoding kit. Sequences were pre-processed, and data were analyzed using WIMP workflow on EPI2ME (ONT) or Minimap2 software with rrn database.Results. Full-length 16S rRNA and the rrn operon retrieved the microbiota composition from the bacterial isolate, the mock communities and the complex skin samples, even at the genus and species level. For Staphylococcus pseudintermedius isolate, when using EPI2ME, the amplicons were assigned to the correct bacterial species in ~98% of the cases with rrn operon as the marker, and ~68% of the cases with 16S rRNA gene respectively. 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, rrn operon would be the best choice.

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Section: Discussionsupporting

confidence: 68%

Section: Introductionmentioning

confidence: 99%

Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and whole rrn operon

Cuscó¹,

Catozzi

Viñes

et al. 2018

Preprint

Self Cite

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“…The full protocol is available on protocols.io . (Cusco et al, 2017;Klindworth et al, 2013). First, we compared 3 fecal samples (run in duplicate) each from wild vs. captive animals of two primate species: the howler monkey (Alouatta seniculus) and the spider monkey (Ateles chamek).…”

Section: Case Studiesmentioning

confidence: 99%

Genomics in the jungle: using portable sequencing as a teaching tool in field courses

Watsa

Pomerantz

et al. 2019

Preprint

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Genetic research is a rapidly evolving field of study that is increasingly being utilized as a tool for wildlife conservation. However, researchers and science educators in remote areas can often find it difficult to access the latest genetic technologies, often due to a combination of high costs, bulky equipment, and lack of infrastructure. Recent technological innovations are resulting in portable, low-cost instruments that enable next-generation sequencing in remote environments, offering new opportunities to generate a more widespread network of trained conservation scientists, particularly within regions of high biodiversity. What is currently lacking are formalized educational efforts to teach participants in biodiverse areas with hands-on training in molecular biology and real-time DNA sequencing techniques. To address this challenge, we report the design and summarized feedback/outcomes of a conservation genetics field course, called 'Genomics in the Jungle', that took place at a field research station in the Amazon rainforest of southeastern Peru. The program was established by a small US-based NGO, Field Projects International, and facilitated by a local eco-tourism company in Peru, Inkaterra. We utilized portable sequencing technologies from Oxford Nanopore Technologies, and in-kind support from the manufacturers MiniPCR, MiniOne Systems, Promega, and New England Biolabs. Participants included a mix of non-Peruvian students and local/regional students, some of which had no prior exposure to a genetics laboratory. Overall, we maintain that portable sequencing technology is democratizing scientific research and conservation efforts, and is a major step forward for science educators and conservationists.

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“…Combined with the diverse kits offered, the ONT is a versatile alternative that can be used for multiple purposes in microbiome studies. There are some publications comparing Illumina and nanopore sequencing (Shin et al, 2016), although most of them focus on 16s rRNA gene sequencing (Cusco et al, 2017; Ma, Stachler, & Bibby, 2017). These previous studies showed a promising potential of nanopore sequencing for species detection with a high correlation between the results obtained by Illumina and ONT at the phylum and genus levels, despite the smaller basecalling accuracy from ONT compared with Illumina.…”

Section: Introductionmentioning

confidence: 99%

“…Moreover, analysis of the 16S rRNA gene is still preferred over shotgun metagenomics using the MinION and most of these studies have been conducted on mouse (Shin et al, 2016) and human gut microbiota (Leggett et al, 2017), proving consistent results for individual taxonomic profiles. Other animal microbiomes have also been sequenced with the MinION, the dog skin microbiota was characterized using a mock community and dog skin samples to test new 16s rRNA primers, finding accurate taxonomic results at the genus level (Cusco et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Long reads from Nanopore sequencing as a tool for animal microbiome studies

Delgado

Serrano

Gonzalez

et al. 2019

Preprint

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19In the era of bioinformatics and metagenomics, the study of the ruminal microbiome has gained considerable 20 relevance in the field of animal breeding, since the composition of the rumen microbiota significantly impacts 21 production and the environment. Illumina sequencing is considered the gold standard for the analysis of 22 microbiomes, but it is limited by obtaining only short DNA sequences to analyze. As an alternative, Oxford 23 Nanopore Technologies (ONT) has developed a new sequencing technique based on nanopores that can be 24 carried out in the MinION, a portable device with a low initial cost which long DNA readings can be obtained 25 with. The aim of this study was to compare the performance of both types of sequencing applied to samples of 26 ruminal content using a similar pipeline. The ONT sequencing provided similar results to the Illumina 27 sequencing, although it was able to classify a greater number of readings at the species level, possibly due to 28 the increase in the read size. The results also suggest that, due to the size of the reads, it would be possible to 29 obtain the same amount of information in a smaller number of hours. However, detection of archaeal and 30 eukaryotic species is still difficult to accomplish due to their low abundance in the rumen compared to 31 bacteria, suggesting different pipelines and strategies are needed to obtain a whole representation of the less 32 abundant species in the rumen microbiota.33 34 35 36 Background 37 38 The bovine rumen has been studied for years in an attempt to reveal functions and microorganisms associated 39 with nutritional features such as feed efficiency to use them in animal breeding programs for cattle (Knapp, 40 Laur, Vadas, Weiss, & Tricarico, 2014). Lately, methane emissions from ruminants are among the main 41 concerns in animal husbandry, given their contribution to global warming. Microbial cultures were essential 42 for the first descriptions of the rumen content but they are usually hard to achieve (Creevey, Kelly, 43 Henderson, & Leahy, 2014). Thanks to the Next Generation Sequencing (NGS) techniques it is now possible 44 to detect different microbial taxa in rumen samples avoiding culture (Seshadri et al., 2018). This has allowed 45 analyzing metagenomic samples in an easier way and to detect non-culturable microbes that broaden the 46 knowledge of complex microbial communities as well as allow quantifying the relative abundances of each 47 65 profiles in municipal sewage, obtaining similar results as Illumina, although it was still necessary to improve 66 the throughput of the sequencing runs. ONT has also been used to detected arbovirus in mosquitos (Batovska, 67 Lynch, Rodoni, Sawbridge, & Cogan, 2017) obtaining comparable results to Illumina sequencing, despite the 68 poorer quality of the reads was poorer. Other studies focus on the applicability of ONT for real-time viral 69 pathogen detection (Greninger et al., 2015) in human blood as it shows similar results to Illumina but provides 70 faster results, which ...

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Using MinION^™to characterize dog skin microbiota through full-length 16S rRNA gene sequencing approach

Cited by 20 publications

References 63 publications

Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and whole rrn operon

Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and whole rrn operon

Genomics in the jungle: using portable sequencing as a teaching tool in field courses

Long reads from Nanopore sequencing as a tool for animal microbiome studies

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Using MinION™to characterize dog skin microbiota through full-length 16S rRNA gene sequencing approach

Cited by 20 publications

References 63 publications

Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and whole rrn operon

Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and whole rrn operon

Genomics in the jungle: using portable sequencing as a teaching tool in field courses

Long reads from Nanopore sequencing as a tool for animal microbiome studies

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Product

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Using MinION^™to characterize dog skin microbiota through full-length 16S rRNA gene sequencing approach