Multi-locus and long amplicon sequencing approach to study microbial diversity at species level using the MinION™ portable nanopore sequencer

Benı́tez-Páez, Alfonso; Sanz, Yolanda

doi:10.1101/117143

Cited by 9 publications

(12 citation statements)

References 34 publications

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“…However, portable sequencing platform, which enables real-time analysis pipelines (Lu et al, 2016). This platform has been broadly applied over the last few years, especially for testing their suitability for in-field or clinical applications (Pomerantz et al, 2018;Orsini et al, 2018), but also for sequencing complete prokaryotic and eukaryotic genomes (Loman et al, 2015;Wick et al, 2017;Deschamps et al, 2018;Jain et al, 2018) and for characterizing microbial communities (Hardegen et al, 2018;Benítez-Páez and Sanz, 2017).…”

Section: Introductionmentioning

confidence: 99%

Assembly methods for nanopore-based metagenomic sequencing: a comparative study

Latorre-Pérez¹,

Villalba-Bermell²,

Pascual³

et al. 2019

Preprint

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BackgroundMetagenomic sequencing has lead to the recovery of previously unexplored microbial genomes. In this sense, short-reads sequencing platforms often result in highly fragmented metagenomes, thus complicating downstream analyses. Third generation sequencing technologies, such as MinION, could lead to more contiguous assemblies due to their ability to generate long reads. Nevertheless, there is a lack of studies evaluating the suitability of the available assembly tools for this new type of data.FindingsWe benchmarked the ability of different short-reads and long-reads tools to assembly two different commercially available mock communities, and observed remarkable differences in the resulting assemblies depending on the software of choice. Short-reads metagenomic assemblers proved unsuitable for MinION data. Among the long-reads assemblers tested, Flye and Canu were the only ones performing well in all the datasets. These tools were able to retrieve complete individual genomes directly from the metagenome, and assembled a bacterial genome in only two contigs in the best scenario. Despite the intrinsic high error of long-reads technologies, Canu and Flye lead to high accurate assemblies (~99.4-99.8 % of accuracy). However, errors still had an impact on the prediction of biosynthetic gene clusters.ConclusionsMinION metagenomic sequencing data proved sufficient for assembling low-complex microbial communities, leading to the recovery of highly complete and contiguous individual genomes. This work is the first systematic evaluation of the performance of different assembly tools on MinION data, and may help other researchers willing to use this technology to choose the most appropriate software depending on their goals. Future work is still needed in order to assess the performance of Oxford Nanopore MinION data on more complex microbiomes.

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Section: Introductionmentioning

confidence: 99%

Assembly methods for nanopore-based metagenomic sequencing: a comparative study

Latorre-Pérez¹,

Villalba-Bermell²,

Pascual³

et al. 2019

Preprint

View full text Add to dashboard Cite

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“…In contrast, emerging third-generation sequencing technologies -MinION (Oxford Nanopore Technologies; https://nanoporetech.com/) and PacBio SMRT sequencing (Pacific Biosciences; http://www.pacb.com/) -offer the possibility to sequence long DNA amplicons in a single read, very much like Sanger does so well for short amplicons. Both of these technologies are suitable for high-accuracy, long-range sequencing (Singer et al 2016;Benitez-Paez and Sanz 2017;Tedersoo et al 2018;Karst et al 2018). PacBio excels where high accuracy is needed due to its circular consensus sequencing mode.…”

Section: Introductionmentioning

confidence: 99%

Introducing ribosomal tandem repeat barcoding for fungi

Wurzbacher

Larsson

Bengtsson‐Palme

et al. 2018

Preprint

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Sequence analysis of the various ribosomal genetic markers is the dominant molecular method for identification and description of fungi. However, there is little agreement on what ribosomal markers should be used, and research groups utilize different markers depending on what fungal groups are targeted. New environmental fungal lineages known only from DNA data reveal significant gaps in the coverage of the fungal kingdom both in terms of taxonomy and marker coverage in the reference sequence databases. In order to integrate references covering all of the ribosomal markers, we present three sets of general primers that allow the amplification of the complete ribosomal operon from the ribosomal tandem repeats. The primers cover all ribosomal markers (ETS, SSU, ITS1, 5.8S, ITS2, LSU, and IGS) from the 5' end of the ribosomal operon all the way to the 3' end. We coupled these primers successfully with third generation sequencing (PacBio and Nanopore sequencing) to showcase our approach on authentic fungal herbarium specimens. In particular, we were able to generate high-quality reference data with Nanopore sequencing in a high-throughput manner, showing that the generation of reference data can be achieved on a regular desktop computer without the need for a large-scale sequencing facility. The quality of the Nanopore generated sequences was 99.85 %, which is comparable with the 99.78 % accuracy described for Sanger sequencing. With this work, we hope to stimulate the generation of a new comprehensive standard of ribosomal reference data with the ultimate aim to close the huge gaps in our reference datasets.

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“…Besides, it can provide a rapid real-time and on-demand analysis very useful on clinical applications. Several studies targeting the full 16S rRNA gene have already been performed using MinION TM to: i) identify pure bacterial culture (31); ii) characterize artificial and alreadycharacterized bacterial communities (mock community) (32)(33)(34); and to iii) characterize complex microbiota samples, from mouse gut (35), wastewater (31) and pleural effusion from a patient with empyema (34).…”

Section: Introductionmentioning

confidence: 99%

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

Cuscó

Viñes

D’Andreano

et al. 2017

Preprint

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The most common strategy to assess microbiota is sequencing specific hypervariable regions of 16S rRNA gene using 2 nd generation platforms (such as MiSeq or Ion Torrent PGM). Despite obtaining high-quality reads, many sequences fail to be classified at the genus or species levels due to their short length. This pitfall can be overcome sequencing the full-length 16S rRNA gene (1,500bp) by 3 rd generation sequencers.We aimed to assess the performance of nanopore sequencing using MinION™ on characterizing microbiota complex samples. First set-up step was performed using a staggered mock community (HM-783D). Then, we sequenced a pool of several dog skin microbiota samples previously sequenced by Ion Torrent PGM. Sequences obtained for full-length 16S rRNA with degenerated primers retrieved increased richness estimates at high taxonomic level (Bacteria and Archaea) that were missed with short-reads. Besides, we were able to obtain taxonomic assignments down to species level, although it was not always feasible due to: i) incomplete database; ii) primer set chosen; iii) low taxonomic resolution of 16S rRNA gene within some genera; and/or iv) sequencing errors. Nanopore sequencing of the full-length 16S rRNA gene using MinION™ with 1D sequencing kit allowed us inferring microbiota composition of a complex microbial community to lower taxonomic levels than short-reads from 2 nd generation sequencers.

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Multi-locus and long amplicon sequencing approach to study microbial diversity at species level using the MinION™ portable nanopore sequencer

Abstract: Abstract

Cited by 9 publications

References 34 publications

Assembly methods for nanopore-based metagenomic sequencing: a comparative study

Assembly methods for nanopore-based metagenomic sequencing: a comparative study

Introducing ribosomal tandem repeat barcoding for fungi

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

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Multi-locus and long amplicon sequencing approach to study microbial diversity at species level using the MinION™ portable nanopore sequencer

Abstract: Abstract

Cited by 9 publications

References 34 publications

Assembly methods for nanopore-based metagenomic sequencing: a comparative study

Assembly methods for nanopore-based metagenomic sequencing: a comparative study

Introducing ribosomal tandem repeat barcoding for fungi

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

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Product

Resources

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