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

2017 ◽  
Author(s):  
Anna Cuscó ◽  
Joaquim Viñes ◽  
Sara D’Andreano ◽  
Francesca Riva ◽  
Joaquim Casellas ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Full Length ◽  
Taxonomic Resolution ◽  
Rrna Gene ◽  
Nanopore Sequencing ◽  
Ion Torrent ◽  
Skin Microbiota ◽  
Short Reads ◽  
Ion Torrent Pgm

AbstractThe most common strategy to assess microbiota is sequencing specific hypervariable regions of 16S rRNA gene using 2ndgeneration 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 3rdgeneration 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 2ndgeneration sequencers.

scholarly journals Establishment and Assessment of An Amplicon Sequencing Method Targeting The 16S-ITS-23S rRNA Operon For Analysis of The Equine Gut Microbiome

2021 ◽  
Author(s):  
Yuta Kinoshita ◽  
Hidekazu NIWA ◽  
Eri UCHIDA-FUJII ◽  
Toshio NUKADA
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Amplicon Sequencing ◽  
Full Length ◽  
Rrna Operon ◽  
Rrna Genes ◽  
Taxonomic Resolution ◽  
23S Rrna ◽  
Rrna Gene ◽  
Fecal Samples

Abstract Microbial communities are commonly studied by using amplicon sequencing of part of the 16S rRNA gene. Sequencing of the full-length 16S rRNA gene can provide higher taxonomic resolution and accuracy. To obtain even higher taxonomic resolution, with as few false-positives as possible, we assessed a method using long amplicon sequencing targeting the rRNA operon combined with a CCMetagen pipeline. Taxonomic assignment had >90% accuracy at the species level in a mock sample and at the family level in equine fecal samples, generating similar taxonomic composition as shotgun sequencing. The rRNA operon amplicon sequencing of equine fecal samples underestimated compositional percentages of bacterial strains containing unlinked rRNA genes by a third to almost a half, but unlinked rRNA genes had a limited effect on the overall results. The rRNA operon amplicon sequencing with the A519F + U2428R primer set was able to reflect archaeal genomes, whereas full-length 16S rRNA with 27F + 1492R could not. Therefore, we conclude that amplicon sequencing targeting the rRNA operon captures more detailed variations of bacterial and archaeal microbiota.

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

2018 ◽  
Author(s):  
Anna Cusco ◽  
Carlotta Catozzi ◽  
Joaquim Vines ◽  
Armand Sanchez ◽  
Olga Francino
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Species Level ◽  
Full Length ◽  
23S Rrna ◽  
Rrna Gene ◽  
Nanopore Sequencing ◽  
Microbiota Composition ◽  
Staphylococcus Pseudintermedius ◽  
Mock Communities

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 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 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 low-biomass 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 the 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.

scholarly journals NanoAmpli-Seq: A workflow for amplicon sequencing for mixed microbial communities on the nanopore sequencing platform

2018 ◽  
Author(s):  
Szymon T Calus ◽  
Umer Z Ijaz ◽  
Ameet J Pinto
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Amplicon Sequencing ◽  
Error Rates ◽  
Full Length ◽  
Rrna Gene ◽  
Nanopore Sequencing ◽  
Short Read ◽  
Sequencing Platform ◽  
Sequencing Platforms

AbstractBackgroundAmplicon sequencing on Illumina sequencing platforms leverages their deep sequencing and multiplexing capacity, but is limited in genetic resolution due to short read lengths. While Oxford Nanopore or Pacific Biosciences platforms overcome this limitation, their application has been limited due to higher error rates or smaller data output.ResultsIn this study, we introduce an amplicon sequencing workflow, i.e., NanoAmpli-Seq, that builds on Intramolecular-ligated Nanopore Consensus Sequencing (INC-Seq) approach and demonstrate its application for full-length 16S rRNA gene sequencing. NanoAmpli-Seq includes vital improvements to the aforementioned protocol that reduces sample-processing time while significantly improving sequence accuracy. The developed protocol includes chopSeq software for fragmentation and read orientation correction of INC-Seq consensus reads while nanoClust algorithm was designed for read partitioning-based de novo clustering and within cluster consensus calling to obtain full-length 16S rRNA gene sequences.ConclusionsNanoAmpli-Seq accurately estimates the diversity of tested mock communities with average sequence accuracy of 99.5% for 2D and 1D2 sequencing on the nanopore sequencing platform. Nearly all residual errors in NanoAmpli-Seq sequences originate from deletions in homopolymer regions, indicating that homopolymer aware basecalling or error correction may allow for sequencing accuracy comparable to short-read sequencing platforms.

scholarly journals Evaluation of 16S rRNA gene sequencing for species and strain-level microbiome analysis

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Jethro S. Johnson ◽  
Daniel J. Spakowicz ◽  
Bo-Young Hong ◽  
Lauren M. Petersen ◽  
Patrick Demkowicz ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput Sequencing ◽  
Full Length ◽  
Strain Level ◽  
Taxonomic Resolution ◽  
Rrna Gene ◽  
Variable Regions ◽  
16S Gene ◽  
Sequencing Platforms

Abstract The 16S rRNA gene has been a mainstay of sequence-based bacterial analysis for decades. However, high-throughput sequencing of the full gene has only recently become a realistic prospect. Here, we use in silico and sequence-based experiments to critically re-evaluate the potential of the 16S gene to provide taxonomic resolution at species and strain level. We demonstrate that targeting of 16S variable regions with short-read sequencing platforms cannot achieve the taxonomic resolution afforded by sequencing the entire (~1500 bp) gene. We further demonstrate that full-length sequencing platforms are sufficiently accurate to resolve subtle nucleotide substitutions (but not insertions/deletions) that exist between intragenomic copies of the 16S gene. In consequence, we argue that modern analysis approaches must necessarily account for intragenomic variation between 16S gene copies. In particular, we demonstrate that appropriate treatment of full-length 16S intragenomic copy variants has the potential to provide taxonomic resolution of bacterial communities at species and strain level.

scholarly journals Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and the 16S-ITS-23S of the rrn operon

F1000Research ◽  
2019 ◽  
Vol 7 ◽  
pp. 1755 ◽  
Author(s):  
Anna Cuscó ◽  
Carlotta Catozzi ◽  
Joaquim Viñes ◽  
Armand Sanchez ◽  
Olga Francino
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Genetic Marker ◽  
Species Level ◽  
Full Length ◽  
Rrna Gene ◽  
Nanopore Sequencing ◽  
Microbiota Composition ◽  
The 16S Rrna Gene ◽  
Mock Communities

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.

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

F1000Research ◽  
2018 ◽  
Vol 7 ◽  
pp. 1755 ◽  
Author(s):  
Anna Cuscó ◽  
Carlotta Catozzi ◽  
Joaquim Viñes ◽  
Armand Sanchez ◽  
Olga Francino
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Species Level ◽  
Full Length ◽  
Rrna Gene ◽  
Nanopore Sequencing ◽  
Microbiota Composition ◽  
Staphylococcus Pseudintermedius ◽  
The 16S Rrna Gene ◽  
Mock Communities

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.

scholarly journals Establishment and assessment of an amplicon sequencing method targeting the 16S-ITS-23S rRNA operon for analysis of the equine gut microbiome

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuta Kinoshita ◽  
Hidekazu Niwa ◽  
Eri Uchida-Fujii ◽  
Toshio Nukada
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Amplicon Sequencing ◽  
Full Length ◽  
Rrna Operon ◽  
Rrna Genes ◽  
Taxonomic Resolution ◽  
23S Rrna ◽  
Rrna Gene ◽  
Fecal Samples

AbstractMicrobial communities are commonly studied by using amplicon sequencing of part of the 16S rRNA gene. Sequencing of the full-length 16S rRNA gene can provide higher taxonomic resolution and accuracy. To obtain even higher taxonomic resolution, with as few false-positives as possible, we assessed a method using long amplicon sequencing targeting the rRNA operon combined with a CCMetagen pipeline. Taxonomic assignment had > 90% accuracy at the species level in a mock sample and at the family level in equine fecal samples, generating similar taxonomic composition as shotgun sequencing. The rRNA operon amplicon sequencing of equine fecal samples underestimated compositional percentages of bacterial strains containing unlinked rRNA genes by a fourth to a third, but unlinked rRNA genes had a limited effect on the overall results. The rRNA operon amplicon sequencing with the A519F + U2428R primer set was able to detect some kind of archaeal genomes such as Methanobacteriales and Methanomicrobiales, whereas full-length 16S rRNA with 27F + 1492R could not. Therefore, we conclude that amplicon sequencing targeting the rRNA operon captures more detailed variations of equine microbiota.

scholarly journals A method for high precision sequencing of near full-length 16S rRNA genes on an Illumina MiSeq

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2492 ◽  
Author(s):  
Catherine M. Burke ◽  
Aaron E. Darling
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
Single Molecule ◽  
Illumina Miseq ◽  
Full Length ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Bacterial Taxonomy

BackgroundThe bacterial 16S rRNA gene has historically been used in defining bacterial taxonomy and phylogeny. However, there are currently no high-throughput methods to sequence full-length 16S rRNA genes present in a sample with precision.ResultsWe describe a method for sequencing near full-length 16S rRNA gene amplicons using the high throughput Illumina MiSeq platform and test it using DNA from human skin swab samples. Proof of principle of the approach is demonstrated, with the generation of 1,604 sequences greater than 1,300 nt from a single Nano MiSeq run, with accuracy estimated to be 100-fold higher than standard Illumina reads. The reads were chimera filtered using information from a single molecule dual tagging scheme that boosts the signal available for chimera detection.ConclusionsThis method could be scaled up to generate many thousands of sequences per MiSeq run and could be applied to other sequencing platforms. This has great potential for populating databases with high quality, near full-length 16S rRNA gene sequences from under-represented taxa and environments and facilitates analyses of microbial communities at higher resolution.

scholarly journals High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution

2019 ◽  
Vol 47 (18) ◽  
pp. e103-e103 ◽  
Author(s):  
Benjamin J Callahan ◽  
Joan Wong ◽  
Cheryl Heiner ◽  
Steve Oh ◽  
Casey M Theriot ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
Amplicon Sequencing ◽  
Full Length ◽  
Rrna Gene ◽  
Single Nucleotide ◽  
Full Complement ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

AbstractTargeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed Escherichia coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains. There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use.

Superior resolution characterisation of microbial diversity in anaerobic digesters using full-length 16S rRNA gene amplicon sequencing

2020 ◽  
Vol 178 ◽  
pp. 115815 ◽  
Author(s):  
Theo Y.C. Lam ◽  
Ran Mei ◽  
Zhuoying Wu ◽  
Patrick K.H. Lee ◽  
Wen-Tso Liu ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Diversity ◽  
Amplicon Sequencing ◽  
Full Length ◽  
Rrna Gene ◽  
Anaerobic Digesters
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