scholarly journals Full-Length 16S rRNA and ITS Gene Sequencing Revealed Rich Microbial Flora in Roots of Cycas spp. in China

2021 ◽  
Vol 17 ◽  
pp. 117693432198971
Author(s):  
Melissa H Pecundo ◽  
Aimee Caye G Chang ◽  
Tao Chen ◽  
Thomas Edison E dela Cruz ◽  
Hai Ren ◽  
...  
Keyword(s):  
Community Structure ◽  
16S Rrna ◽  
Current Knowledge ◽  
Its Region ◽  
Amplicon Sequencing ◽  
Full Length ◽  
Ex Situ ◽  
Rrna Gene ◽  
Coralloid Roots ◽  
Different Strains

Cycads have developed a complex root system categorized either as normal or coralloid roots. Past literatures revealed that a great diversity of key microbes is associated with these roots. This recent study aims to comprehensively determine the diversity and community structure of bacteria and fungi associated with the roots of two Cycas spp. endemic to China, Cycas debaoensis Zhong & Chen and Cycas fairylakea D.Y. Wang using high-throughput amplicon sequencing of the full-length 16S rRNA (V1-V9 hypervariable) and short fragment ITS region. The total DNA from 12 root samples were extracted, amplified, sequenced, and analyzed. Resulting sequences were clustered into 61 bacteria and 2128 fungal OTUs. Analysis of community structure revealed that the coralloid roots were dominated mostly by the nitrogen-fixer Nostocaceae but also contain other non-diazotrophic bacteria. The sequencing of entire 16S rRNA gene identified four different strains of cyanobacteria under the heterocystous genera Nostoc and Desmonostoc. Meanwhile, the top bacterial families in normal roots were Xanthobacteraceae, Burkholderiaceae, and Bacillaceae. Moreover, a diverse fungal community was also found in the roots of cycads and the predominating families were Ophiocordycipitaceae, Nectriaceae, Bionectriaceae, and Trichocomaceae. Our results demonstrated that bacterial diversity in normal roots of C. fairylakea is higher in richness and abundance than C. debaoensis. On the other hand, a slight difference, albeit insignificant, was noted for the diversity of fungi among root types and host species as the number of shared taxa is relatively high (67%). Our results suggested that diverse microbes are present in roots of cycads which potentially interact together to support cycads survival. Our study provided additional knowledge on the microbial diversity and composition in cycads and thus expanding our current knowledge on cycad-microbe association. Our study also considered the possible impact of ex situ conservation on cyanobiont community of cycads.

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 Identification of Microbial Profiles in Heavy-Metal-Contaminated Soil from Full-Length 16S rRNA Reads Sequenced by a PacBio System

2019 ◽  
Vol 7 (9) ◽  
pp. 357 ◽  
Author(s):  
Moonsuk Hur ◽  
Soo-Je Park
Keyword(s):  
Heavy Metals ◽  
Heavy Metal ◽  
Community Structure ◽  
16S Rrna ◽  
Microbial Communities ◽  
Contaminated Soil ◽  
Contaminated Soils ◽  
Full Length ◽  
Rrna Gene ◽  
Microbial Composition

Heavy metal pollution is a serious environmental problem as it adversely affects crop production and human activity. In addition, the microbial community structure and composition are altered in heavy-metal-contaminated soils. In this study, using full-length 16S rRNA gene sequences obtained by a PacBio RS II system, we determined the microbial diversity and community structure in heavy-metal-contaminated soil. Furthermore, we investigated the microbial distribution, inferred their putative functional traits, and analyzed the environmental effects on the microbial compositions. The soil samples selected in this study were heavily and continuously contaminated with various heavy metals due to closed mines. We found that certain microorganisms (e.g., sulfur or iron oxidizers) play an important role in the biogeochemical cycle. Using phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis, we predicted Kyoto Encyclopedia of Genes and Genomes (KEGG) functional categories from abundances of microbial communities and revealed a high proportion belonging to transport, energy metabolism, and xenobiotic degradation in the studied sites. In addition, through full-length analysis, Conexibacter-like sequences, commonly identified by environmental metagenomics among the rare biosphere, were detected. In addition to microbial composition, we confirmed that environmental factors, including heavy metals, affect the microbial communities. Unexpectedly, among these environmental parameters, electrical conductivity (EC) might have more importance than other factors in a community description analysis.

scholarly journals Unique chemical parameters and microbial activity lead to increased archaeological preservation at the Roman frontier site of Vindolanda, UK

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
C. H. Orr ◽  
R. Williams ◽  
H. H. Halldórsdóttir ◽  
A. Birley ◽  
E. Greene ◽  
...  
Keyword(s):  
Community Structure ◽  
16S Rrna ◽  
Amplicon Sequencing ◽  
Chemical Degradation ◽  
Rrna Gene ◽  
Degradation Mechanisms ◽  
Chemical Parameters ◽  
Anaerobic Conditions ◽  
Archaeological Preservation ◽  
Unique Chemical

AbstractWaterlogged burial conditions impact upon artefact preservation. One major determinant of preservation is presence and behaviour of microorganisms, however, unravelling the mechanisms, especially in waterlogged conditions is challenging. In this study, we analysed elemental composition, bacterial diversity and community structure from excavation trenches at the Roman Site of Vindolanda, Northumberland, UK, using pXRF and 16S rRNA gene amplicon sequencing. Excavation trenches provide information of different occupation periods. The results indicated that microbial communities were dominated by Firmicutes, Bacteroidetes and Proteobacteria at a phylum level. Samples which also had visible vivianite presence showed that there were marked increases in Methylophilus. Methylophilus might be associated with favourable preservation in these anaerobic conditions. More research is needed to clearly link the presence of Methylophilus with vivianite production. The study emphasises the need for further integration of chemical and microbiome approaches, especially in good preservation areas, to explore microbial and chemical degradation mechanisms.

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

2018 ◽  
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 allowedE. colistrains 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 severalE. colistrains.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.

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 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.

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