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 Erratum for Salipante et al., Performance Comparison of Illumina and Ion Torrent Next-Generation Sequencing Platforms for 16S rRNA-Based Bacterial Community Profiling

2016 ◽  
Vol 82 (14) ◽  
pp. 4453-4453 ◽  
Author(s):  
Stephen J. Salipante ◽  
Toana Kawashima ◽  
Christopher Rosenthal ◽  
Daniel R. Hoogestraat ◽  
Lisa A. Cummings ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Bacterial Community ◽  
16S Rrna ◽  
Performance Comparison ◽  
Ion Torrent ◽  
Next Generation ◽  
Community Profiling ◽  
Sequencing Platforms ◽  
Generation Sequencing

scholarly journals Bacterial Community Shift in Treated Periodontitis Patients Revealed by Ion Torrent 16S rRNA Gene Amplicon Sequencing

PLoS ONE ◽  
2012 ◽  
Vol 7 (8) ◽  
pp. e41606 ◽  
Author(s):  
Sebastian Jünemann ◽  
Karola Prior ◽  
Rafael Szczepanowski ◽  
Inga Harks ◽  
Benjamin Ehmke ◽  
...  
Keyword(s):  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Ion Torrent ◽  
Community Shift

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 A converging subset of soil bacterial taxa is permissive to the IncP-1 plasmid pKJK5 across a range of soil copper contamination

2020 ◽  
Vol 96 (11) ◽  
Author(s):  
Jianxiao Song ◽  
Uli Klümper ◽  
Leise Riber ◽  
Arnaud Dechesne ◽  
Barth F Smets ◽  
...  
Keyword(s):  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Sequence Similarity ◽  
Amplicon Sequencing ◽  
Plasmid Transfer ◽  
Rrna Gene ◽  
Long Term Effects ◽  
Cu Contamination ◽  
Soil Bacterial

ABSTRACT Stressors like metals or antibiotics can affect bacterial community permissiveness for plasmid uptake, but there is little knowledge about long-term effects of such stressors on the evolution of community permissiveness. We assessed the effect of more than 90 years of soil Cu contamination on bacterial community permissiveness (i.e. uptake ability) toward a gfp-tagged IncP-1 plasmid (pKJK5) introduced via an Escherichia coli donor. Plasmid transfer events from the donor to the recipient soil bacterial community were quantified and transconjugants were subsequently isolated by fluorescence activated cell sorting and identified by 16S rRNA gene amplicon sequencing. Transfer frequency of plasmid pKJK5 was reduced in bacterial communities extracted from highly Cu contaminated (4526 mg kg−1) soil compared to corresponding communities extracted from moderately (458 mg kg−1) Cu contaminated soil and a low Cu reference soil (15 mg kg−1). The taxonomic composition of the transconjugal pools showed remarkable similarities irrespective of the degree of soil Cu contamination and despite contrasting compositions of the extracted recipient communities and the original soil communities. Permissiveness assessed at the level of individual operational taxonomic units (OTUs; 16S rRNA gene 97% sequence similarity threshold) was only slightly affected by soil Cu level and high replicate variability of OTU-level permissiveness indicated a role of stochastic events in IncP-1 plasmid transfer or strain-to-strain permissiveness variability.

Influence of sampling and DNA extraction on 16S rRNA gene amplicon sequencing - Comparison of the bacterial community between two food processing plants

LWT ◽  
2018 ◽  
Vol 90 ◽  
pp. 186-192 ◽  
Author(s):  
Anna Kristina Witte ◽  
Christine Leeb ◽  
Beate Pinior ◽  
Patrick Mester ◽  
Susanne Fister ◽  
...  
Keyword(s):  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Extraction ◽  
Food Processing ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Processing Plants

scholarly journals Profiling acetogenic community dynamics in anaerobic digesters - comparative analyses using next-generation sequencing and T-RFLP

2021 ◽  
Author(s):  
Abhijeet Singh ◽  
Bettina Müller ◽  
Anna Schnürer
Keyword(s):  
Bacterial Community ◽  
16S Rrna ◽  
High Throughput ◽  
Community Dynamics ◽  
Temporal Dynamics ◽  
Gene Sequencing ◽  
Process Efficiency ◽  
Rrna Gene ◽  
Anaerobic Digesters ◽  
Community Profiling

ABSTRACTAcetogens play a key role in anaerobic degradation of organic material and in maintaining biogas process efficiency. Profiling this community and its temporal changes can help evaluate process stability and function, especially under disturbance/stress conditions, and avoid complete process failure. The formyltetrahydrofolate synthetase (FTHFS) gene can be used as a marker for acetogenic community profiling in diverse environments. In this study, we developed a new high-throughput FTHFS gene sequencing method for acetogenic community profiling and compared it with conventional T-RFLP of the FTHFS gene, 16S rRNA gene-based profiling of the whole bacterial community, and indirect analysis via 16S rRNA profiling of the FTHFS gene-harbouring community. Analyses and method comparisons were made using samples from two laboratory-scale biogas processes, one operated under stable control and one exposed to controlled overloading disturbance. Comparative analysis revealed satisfactory detection of the bacterial community and its changes for all methods, but with some differences in resolution and taxonomic identification. FTHFS gene sequencing was found to be the most suitable and reliable method to study acetogenic communities. These results pave the way for community profiling in various biogas processes and in other environments where the dynamics of acetogenic bacteria have not been well studied.GRAPHICAL ABSTRACTONE SENTENCE SUMMARYOur high-throughput FTHFS gene AmpSeq method for barcoded samples and unsupervised analysis with AcetoScan accurately reveals temporal dynamics of acetogenic community structure in anaerobic digesters.

scholarly journals High-Resolution Melt Analysis for Rapid Comparison of Bacterial Community Compositions

2014 ◽  
Vol 80 (12) ◽  
pp. 3568-3575 ◽  
Author(s):  
Mathis Hjort Hjelmsø ◽  
Lars Hestbjerg Hansen ◽  
Jacob Bælum ◽  
Louise Feld ◽  
William E. Holben ◽  
...  
Keyword(s):  
High Resolution ◽  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Community Composition ◽  
Bacterial Community Composition ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
High Resolution Melt ◽  
Hrm Analysis

ABSTRACTIn the study of bacterial community composition, 16S rRNA gene amplicon sequencing is today among the preferred methods of analysis. The cost of nucleotide sequence analysis, including requisite computational and bioinformatic steps, however, takes up a large part of many research budgets. High-resolution melt (HRM) analysis is the study of the melt behavior of specific PCR products. Here we describe a novel high-throughput approach in which we used HRM analysis targeting the 16S rRNA gene to rapidly screen multiple complex samples for differences in bacterial community composition. We hypothesized that HRM analysis of amplified 16S rRNA genes from a soil ecosystem could be used as a screening tool to identify changes in bacterial community structure. This hypothesis was tested using a soil microcosm setup exposed to a total of six treatments representing different combinations of pesticide and fertilization treatments. The HRM analysis identified a shift in the bacterial community composition in two of the treatments, both including the soil fumigant Basamid GR. These results were confirmed with both denaturing gradient gel electrophoresis (DGGE) analysis and 454-based 16S rRNA gene amplicon sequencing. HRM analysis was shown to be a fast, high-throughput technique that can serve as an effective alternative to gel-based screening methods to monitor microbial community composition.

scholarly journals Characterization of bacterial community shift in human Ulcerative Colitis patients revealed by Illumina based 16S rRNA gene amplicon sequencing

Gut Pathogens ◽  
2014 ◽  
Vol 6 (1) ◽  
pp. 22 ◽  
Author(s):  
Sandeep A Walujkar ◽  
Dhiraj P Dhotre ◽  
Nachiket P Marathe ◽  
Parimal S Lawate ◽  
Renu S Bharadwaj ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Community Shift

scholarly journals Engineering CRISPR/Cas9 to mitigate abundant host contamination for 16S rRNA gene-based amplicon sequencing

Microbiome ◽  
2020 ◽  
Vol 8 (1) ◽  
Author(s):  
Luyang Song ◽  
Kabin Xie
Keyword(s):  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput Sequencing ◽  
Bacterial Species ◽  
Amplicon Sequencing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Plant Microbiota

Abstract Background High-throughput sequencing of bacterial 16S rRNA gene (16S-seq) is a useful and common method for studying bacterial community structures. However, contamination of the 16S rRNA genes from the mitochondrion and plastid hinders the sensitive bacterial 16S-seq in plant microbiota profiling, especially for some plant species such as rice. To date, efficiently mitigating such host contamination without a bias is challenging in 16S rRNA gene-based amplicon sequencing. Results We developed Cas-16S-seq method to reduce abundant host contamination for plant microbiota profiling. This method utilizes the Cas9 nuclease and specific guide RNA (gRNA) to cut 16S rRNA targets during library construction, thereby removing host contamination in 16S-seq. We used rice as an example to validate the feasibility and effectiveness of Cas-16S-seq. We established a bioinformatics pipeline to design gRNAs that specifically target rice 16S rRNA genes without bacterial 16S rRNA off-targets. We compared the effectiveness of Cas-16S-seq with that of the commonly used 16S-seq method for artificially mixed 16S rRNA gene communities, paddy soil, rice root, and phyllosphere samples. The results showed that Cas-16S-seq substantially reduces the fraction of rice 16S rRNA gene sequences from 63.2 to 2.9% in root samples and from 99.4 to 11.6% in phyllosphere samples on average. Consequently, Cas-16S-seq detected more bacterial species than the 16S-seq in plant samples. Importantly, when analyzing soil samples, Cas-16S-seq and 16S-seq showed almost identical bacterial communities, suggesting that Cas-16S-seq with host-specific gRNAs that we designed has no off-target in rice microbiota profiling. Conclusion Our Cas-16S-seq can efficiently remove abundant host contamination without a bias for 16S rRNA gene-based amplicon sequencing, thereby enabling deeper bacterial community profiling with a low cost and high flexibility. Thus, we anticipate that this method would be a useful tool for plant microbiomics.

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.

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