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 Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION™ nanopore sequencing confers species-level resolution

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yoshiyuki Matsuo ◽  
Shinnosuke Komiya ◽  
Yoshiaki Yasumizu ◽  
Yuki Yasuoka ◽  
Katsura Mizushima ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Amplicon Sequencing ◽  
Species Level ◽  
Full Length ◽  
Rrna Gene ◽  
Short Read ◽  
Short Read Sequencing ◽  
Long Read

Abstract Background Species-level genetic characterization of complex bacterial communities has important clinical applications in both diagnosis and treatment. Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene has proven to be a powerful strategy for the taxonomic classification of bacteria. This study aims to improve the method for full-length 16S rRNA gene analysis using the nanopore long-read sequencer MinION™. We compared it to the conventional short-read sequencing method in both a mock bacterial community and human fecal samples. Results We modified our existing protocol for full-length 16S rRNA gene amplicon sequencing by MinION™. A new strategy for library construction with an optimized primer set overcame PCR-associated bias and enabled taxonomic classification across a broad range of bacterial species. We compared the performance of full-length and short-read 16S rRNA gene amplicon sequencing for the characterization of human gut microbiota with a complex bacterial composition. The relative abundance of dominant bacterial genera was highly similar between full-length and short-read sequencing. At the species level, MinION™ long-read sequencing had better resolution for discriminating between members of particular taxa such as Bifidobacterium, allowing an accurate representation of the sample bacterial composition. Conclusions Our present microbiome study, comparing the discriminatory power of full-length and short-read sequencing, clearly illustrated the analytical advantage of sequencing the full-length 16S rRNA gene.

scholarly journals Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION™ nanopore sequencing confers species-level resolution

2020 ◽  
Author(s):  
Yoshiyuki Matsuo ◽  
Shinnosuke Komiya ◽  
Yoshiaki Yasumizu ◽  
Yuki Yasuoka ◽  
Katsura Mizushima ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Amplicon Sequencing ◽  
Species Level ◽  
Full Length ◽  
Rrna Gene ◽  
Short Read ◽  
Short Read Sequencing ◽  
16S Amplicon Sequencing ◽  
Long Read

AbstractBackgroundSpecies-level genetic characterization of complex bacterial communities has important clinical applications in both diagnosis and treatment. Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene has proven to be a powerful strategy for the taxonomic classification of bacteria. This study aims to improve the method for full-length 16S rRNA gene analysis using the nanopore long-read sequencer MinION™. We compared it to the conventional short-read sequencing method in both a mock bacterial community and human fecal samples.ResultsWe modified our existing protocol for full-length 16S amplicon sequencing by MinION™. A new strategy for library construction with an optimized primer set overcame PCR-associated bias and enabled taxonomic classification across a broad range of bacterial species. We compared the performance of full-length and short-read 16S amplicon sequencing for the characterization of human gut microbiota with a complex bacterial composition. The relative abundance of dominant bacterial genera was highly similar between full-length and short-read sequencing. At the species level, MinION™ long-read sequencing had better resolution for discriminating between members of particular taxa such as Bifidobacterium, allowing an accurate representation of the sample bacterial composition.ConclusionsOur present microbiome study, comparing the discriminatory power of full-length and short-read sequencing, clearly illustrated the analytical advantage of sequencing the full-length 16S rRNA gene, which provided the requisite species-level resolution and accuracy in clinical settings.

scholarly journals In-field metagenome and 16S rRNA gene amplicon nanopore sequencing robustly characterize glacier microbiota

2016 ◽  
Author(s):  
Arwyn Edwards ◽  
Aliyah R. Debbonaire ◽  
Samuel M. Nicholls ◽  
Sara M.E. Rassner ◽  
Birgit Sattler ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Greenland Ice Sheet ◽  
Environmental Dna ◽  
Amplicon Sequencing ◽  
Taxonomic Composition ◽  
Rrna Gene ◽  
Nanopore Sequencing ◽  
Nanopore Dna Sequencing ◽  
Sequencing Platforms

ABSTRACTIn the field of observation, chance favours only the prepared mind (Pasteur). Impressive developments in genomics have led microbiology to its third “Golden Age”. However, conventional metagenomics strategies necessitate retrograde transfer of samples from extreme or remote environments for later analysis, rendering the powerful insights gained retrospective in nature, striking a contrast with Pasteur’s dictum. Here we implement highly portable USB-based nanopore DNA sequencing platforms coupled with field-adapted environmental DNA extraction, rapid sequence library generation and off-line analyses of shotgun metagenome and 16S ribosomal RNA gene amplicon profiles to characterize microbiota dwelling within cryoconite holes upon Svalbard glaciers, the Greenland Ice Sheet and the Austrian Alps. We show in-field nanopore sequencing of metagenomes captures taxonomic composition of supraglacial microbiota, while 16S rRNA Furthermore, comparison of nanopore data with prior 16S rRNA gene V1-V3 pyrosequencing from the same samples, demonstrates strong correlations between profiles obtained from nanopore sequencing and laboratory based sequencing approaches. gene amplicon sequencing resolves bacterial community responses to habitat changes. Finally, we demonstrate the fidelity and sensitivity of in-field sequencing by analysis of mock communities using field protocols. Ultimately, in-field sequencing potentiated by nanopore devices raises the prospect of enhanced agility in exploring Earth’s most remote microbiomes.

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

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 Performance Comparison of Illumina and Ion Torrent Next-Generation Sequencing Platforms for 16S rRNA-Based Bacterial Community Profiling

2014 ◽  
Vol 80 (24) ◽  
pp. 7583-7591 ◽  
Author(s):  
Stephen J. Salipante ◽  
Toana Kawashima ◽  
Christopher Rosenthal ◽  
Daniel R. Hoogestraat ◽  
Lisa A. Cummings ◽  
...  
Keyword(s):  
Bacterial Community ◽  
16S Rrna ◽  
Amplicon Sequencing ◽  
Error Rates ◽  
Personal Genome ◽  
Rrna Gene ◽  
Ion Torrent ◽  
Community Profiling ◽  
Sequencing Platforms ◽  
Ion Torrent Platform

ABSTRACTHigh-throughput sequencing of the taxonomically informative 16S rRNA gene provides a powerful approach for exploring microbial diversity. Here we compare the performances of two common “benchtop” sequencing platforms, Illumina MiSeq and Ion Torrent Personal Genome Machine (PGM), for bacterial community profiling by 16S rRNA (V1-V2) amplicon sequencing. We benchmarked performance by using a 20-organism mock bacterial community and a collection of primary human specimens. We observed comparatively higher error rates with the Ion Torrent platform and report a pattern of premature sequence truncation specific to semiconductor sequencing. Read truncation was dependent on both the directionality of sequencing and the target species, resulting in organism-specific biases in community profiles. We found that these sequencing artifacts could be minimized by using bidirectional amplicon sequencing and an optimized flow order on the Ion Torrent platform. Results of bacterial community profiling performed on the mock community and a collection of 18 human-derived microbiological specimens were generally in good agreement for both platforms; however, in some cases, results differed significantly. Disparities could be attributed to the failure to generate full-length reads for particular organisms on the Ion Torrent platform, organism-dependent differences in sequence error rates affecting classification of certain species, or some combination of these factors. This study demonstrates the potential for differential bias in bacterial community profiles resulting from the choice of sequencing platform alone.

scholarly journals Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system

2016 ◽  
Author(s):  
Patrick D Schloss ◽  
Matthew L Jenior ◽  
Charles C. Koumpouras ◽  
Sarah L Westcott ◽  
Sarah K Highlander
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Sequencing ◽  
Error Rate ◽  
Full Length ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Mock Community ◽  
Sequencing Platforms ◽  
The 16S Rrna Gene

Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina's MiSeq, have allowed researchers to obtain millions of high quality, but short sequences. The result of the added sequencing depth has been significant improvements in experimental design. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT) DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3-V5, V1-V3, V1-V5, V1-V6, and V1-V9 variable regions from within the 16S rRNA gene using DNA from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1-V9 region from 0.69 to 0.027%. This error rate is comparable to what has been observed for the shorter reads generated by 454 and Illumina's MiSeq sequencing platforms. Although the per base sequencing cost is still significantly more than that of MiSeq, the prospect of supplementing reference databases with full-length sequences from organisms below the limit of detection from the Sanger approach is exciting.

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 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.

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