scholarly journals Thousands of primer-free, high-quality, full-length SSU rRNA sequences from all domains of life

2016 ◽  
Author(s):  
Søren M. Karst ◽  
Morten S. Dueholm ◽  
Simon J. McIlroy ◽  
Rasmus H. Kirkegaard ◽  
Per H. Nielsen ◽  
...  
Keyword(s):  
Microbial Diversity ◽  
Ribosomal Rna ◽  
Full Length ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Gene Sequences ◽  
Variable Regions ◽  
Domains Of Life ◽  
Rrna Sequences

AbstractRibosomal RNA (rRNA) genes are the consensus marker for determination of microbial diversity on the planet, invaluable in studies of evolution and, for the past decade, high-throughput sequencing of variable regions of ribosomal RNA genes has become the backbone of most microbial ecology studies. However, the underlying reference databases of full-length rRNA gene sequences are underpopulated, ecosystem skewed1, and subject to primer bias2, which hamper our ability to study the true diversity of ecosystems. Here we present an approach that combines reverse transcription of full-length small subunit (SSU) rRNA genes and synthetic long read sequencing by molecular tagging, to generate primer-free, full-length SSU rRNA gene sequences from all domains of life, with a median raw error rate of 0.17%. We generated thousands of full-length SSU rRNA sequences from five well-studied ecosystems (soil, human gut, fresh water, anaerobic digestion, and activated sludge) and obtained sequences covering all domains of life and the majority of all described phyla. Interestingly, 30% of all bacterial operational taxonomic units were novel, compared to the SILVA database (less than 97% similarity). For the Eukaryotes, the novelty was even larger with 63% of all OTUs representing novel taxa. In addition, 15% of the 18S rRNA OTUs were highly novel sequences with less than 80% similarity to the databases. The generation of primer-free full-length SSU rRNA sequences enabled eco-system specific estimation of primer-bias and, especially for eukaryotes, showed a dramatic discrepancy between the in-silico evaluation and primer-free data generated in this study. The large amount of novel sequences obtained here reaffirms that there is still vast, untapped microbial diversity lacking representatives in the SSU rRNA databases and that there might be more than millions after all1, 3. With our new approach, it is possible to readily expand the rRNA databases by orders of magnitude within a short timeframe. This will, for the first time, enable a broad census of the tree of life.

Myosporidium ladogensis n. comb. in burbot Lota lota from Finland: fine structure and microsporidian taxonomy

2020 ◽  
Vol 139 ◽  
pp. 15-23
Author(s):  
SRM Jones ◽  
H Ahonen ◽  
J Taskinen
Keyword(s):  
Host Cell ◽  
Ribosomal Rna ◽  
Small Subunit ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Cell Cytoplasm ◽  
Lota Lota ◽  
Host Cell Cytoplasm ◽  
Pike Perch ◽  
Rrna Sequences

Infections with microsporidian parasites are described in skeletal muscle of burbot Lota lota from Lake Haukivesi, Finland. Infected myocytes contained spores within sporophorous vesicles (SPVs) in contact with host cell cytoplasm, similar to Pleistophora ladogensis in L. lota and smelt Osmerus eperlanus in western Russia and northern Germany. Analysis of small subunit ribosomal RNA (SSU rRNA) gene sequences indicated identity with Myosporidium spraguei in burbot and pike-perch from this lake. The latter is considered a junior synonym of P. ladogensis. Phylogenetic analysis of SSU rRNA sequences resolved the burbot parasite apart from a clade containing the type species P. typicalis, but together with M. merluccius. The parasite is renamed Myosporidium ladogensis (Voronin, 1978) n. comb. Networks of tubular appendages arising from developing meronts and SPVs were associated with degradation of host cell cytoplasm.

scholarly journals Microbial Community Analysis with Ribosomal Gene Fragments from Shotgun Metagenomes

2015 ◽  
Vol 82 (1) ◽  
pp. 157-166 ◽  
Author(s):  
Jiarong Guo ◽  
James R. Cole ◽  
Qingpeng Zhang ◽  
C. Titus Brown ◽  
James M. Tiedje
Keyword(s):  
Community Analysis ◽  
Rrna Genes ◽  
Metagenomic Data ◽  
Rrna Gene ◽  
Processing Unit ◽  
Metagenomic Sequencing ◽  
Ssu Rrna ◽  
Variable Regions ◽  
Content Type ◽  
Central Processing

ABSTRACTShotgun metagenomic sequencing does not depend on gene-targeted primers or PCR amplification; thus, it is not affected by primer bias or chimeras. However, searching rRNA genes from large shotgun Illumina data sets is computationally expensive, and no approach exists for unsupervised community analysis of small-subunit (SSU) rRNA gene fragments retrieved from shotgun data. We present a pipeline, SSUsearch, to achieve the faster identification of short-subunit rRNA gene fragments and enabled unsupervised community analysis with shotgun data. It also includes classification and copy number correction, and the output can be used by traditional amplicon analysis platforms. Shotgun metagenome data using this pipeline yielded higher diversity estimates than amplicon data but retained the grouping of samples in ordination analyses. We applied this pipeline to soil samples with paired shotgun and amplicon data and confirmed bias againstVerrucomicrobiain a commonly used V6-V8 primer set, as well as discovering likely bias againstActinobacteriaand forVerrucomicrobiain a commonly used V4 primer set. This pipeline can utilize all variable regions in SSU rRNA and also can be applied to large-subunit (LSU) rRNA genes for confirmation of community structure. The pipeline can scale to handle large amounts of soil metagenomic data (5 Gb memory and 5 central processing unit hours to process 38 Gb [1 lane] of trimmed Illumina HiSeq2500 data) and is freely available athttps://github.com/dib-lab/SSUsearchunder a BSD license.

scholarly journals Ribovore: ribosomal RNA sequence analysis for GenBank submissions and database curation

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Alejandro A. Schäffer ◽  
Richard McVeigh ◽  
Barbara Robbertse ◽  
Conrad L. Schoch ◽  
Anjanette Johnston ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Ribosomal Rna ◽  
Dna Sequences ◽  
Software Package ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Rrna Sequence ◽  
Ssu Rrna Gene ◽  
Taxonomic Groups ◽  
Rrna Sequences

Abstract Background The DNA sequences encoding ribosomal RNA genes (rRNAs) are commonly used as markers to identify species, including in metagenomics samples that may combine many organismal communities. The 16S small subunit ribosomal RNA (SSU rRNA) gene is typically used to identify bacterial and archaeal species. The nuclear 18S SSU rRNA gene, and 28S large subunit (LSU) rRNA gene have been used as DNA barcodes and for phylogenetic studies in different eukaryote taxonomic groups. Because of their popularity, the National Center for Biotechnology Information (NCBI) receives a disproportionate number of rRNA sequence submissions and BLAST queries. These sequences vary in quality, length, origin (nuclear, mitochondria, plastid), and organism source and can represent any region of the ribosomal cistron. Results To improve the timely verification of quality, origin and loci boundaries, we developed Ribovore, a software package for sequence analysis of rRNA sequences. The and programs are used to validate incoming sequences of bacterial and archaeal SSU rRNA. The program is used to create high-quality datasets of rRNAs from different taxonomic groups. Key algorithmic steps include comparing candidate sequences against rRNA sequence profile hidden Markov models (HMMs) and covariance models of rRNA sequence and secondary-structure conservation, as well as other tests. Nine freely available rRNA databases created and maintained with Ribovore are used for checking incoming GenBank submissions and used by the browser interface at NCBI. Since 2018, Ribovore has been used to analyze more than 50 million prokaryotic SSU rRNA sequences submitted to GenBank, and to select at least 10,435 fungal rRNA RefSeq records from type material of 8350 taxa. Conclusion Ribovore combines single-sequence and profile-based methods to improve GenBank processing and analysis of rRNA sequences. It is a standalone, portable, and extensible software package for the alignment, classification and validation of rRNA sequences. Researchers planning on submitting SSU rRNA sequences to GenBank are encouraged to download and use Ribovore to analyze their sequences prior to submission to determine which sequences are likely to be automatically accepted into GenBank.

Mycobacterium bourgelatii sp. nov., a rapidly growing, non-chromogenic species isolated from the lymph nodes of cattle

2013 ◽  
Vol 63 (Pt_12) ◽  
pp. 4669-4674 ◽  
Author(s):  
Véronique Guérin-Faublée ◽  
Jean-Pierre Flandrois ◽  
Catherine Pichat ◽  
Maria Laura Boschiroli ◽  
Brigitte Lamy
Keyword(s):  
Phylogenetic Analysis ◽  
Lymph Nodes ◽  
Type Species ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Gene Sequences ◽  
Content Type ◽  
Ssu Rrna Gene ◽  
Link Type

Three independent strains of a rapidly growing, non-chromogenic member of the genus Mycobacterium were isolated from lymph nodes of French cattle. Identification of the isolates was carried out using a polyphasic approach. The nearly complete SSU rRNA gene sequences (>1200 bp) of the strains MLB-A23, MLB-A30 and MLB-A84T were identical. A phylogenetic analysis of these unique SSU rRNA gene sequences showed that these strains were most closely related to Mycobacterium intermedium . Further phylogenetic analysis based on concatenated sequences (2854 bp) of four housekeeping genes (hsp65, rpoB, sodA and tuf), the transfer–messenger RNA (tmRNA) and SSU rRNA genes indicated that these three strains represented a distinct species that shares a common ancestor with M. intermedium . Phylogenetic and phenotypic data strongly indicate that the strains MLB-A23, MLB-A30 and MLB-A84T belong to a novel mycobacterial species for which the name Mycobacterium bourgelatii sp. nov. is proposed. The type strain is MLB-A84T ( = CIP 110557T = DSM 45746T).

Phylogenetic analysis of freshwater fish trypanosomes from Europe using ssu rRNA gene sequences and random amplification of polymorphic DNA

Parasitology ◽  
2004 ◽  
Vol 130 (4) ◽  
pp. 405-412 ◽  
Author(s):  
W. C. GIBSON ◽  
J. LOM ◽  
H. PECKOVÁ ◽  
V. R. FERRIS ◽  
P. B. HAMILTON
Keyword(s):  
Phylogenetic Analysis ◽  
Freshwater Fish ◽  
Genetic Relationships ◽  
Small Subunit ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Gene Sequences ◽  
Ssu Rrna Gene ◽  
Random Amplification

The taxonomy and phylogenetic relationships of fish trypanosomes are uncertain. A collection of 22 cloned trypanosome isolates from 14 species of European freshwater fish and 1 species of African freshwater fish were examined by molecular phylogenetic analysis. The small subunit ribosomal RNA (ssu rRNA) genes of 8 clones were sequenced and compared with ssu rRNA gene sequences from a wider selection of vertebrate trypanosome isolates by phylogenetic analysis. All trypanosomes from freshwater fish fell in a single clade, subdivided into 3 groups. This clade sits within a larger, robust clade containing trypanosomes from marine fish and various amphibious vertebrates. All 22 trypanosome clones were analysed by random amplification of polymorphic DNA. The resulting dendrogram shows 3 groups, which are congruent with the groups identified in the ssu rRNA gene phylogeny. Two of the groups contain the majority of trypanosome isolates and within-group variation is slight. These groups do not separate purported trypanosome species distinguished by morphology or host origin, and thus these criteria do not appear to be reliable guides to genetic relationships among fish trypanosomes. However, we suggest that the 2 groups themselves may represent different species of fish trypanosomes. The polymorphic DNA markers we have identified will facilitate future comparisons of the biology of these 2 groups of fish trypanosomes.

scholarly journals Ribovore: ribosomal RNA sequence analysis for GenBank submissions and database curation

2021 ◽  
Author(s):  
Alejandro A. Schäffer ◽  
Richard McVeigh ◽  
Barbara Robbertse ◽  
Conrad L. Schoch ◽  
Anjanette Johnston ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Ribosomal Rna ◽  
Dna Sequences ◽  
Software Package ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Rrna Sequence ◽  
Ssu Rrna Gene ◽  
Taxonomic Groups ◽  
Rrna Sequences

AbstractBackgroundThe DNA sequences encoding ribosomal RNA genes (rRNAs) are commonly used as markers to identify species, including in metagenomics samples that may combine many organismal communities. The 16S small subunit ribosomal RNA (SSU rRNA) gene is typically used to identify bacterial and archaeal species. The nuclear 18S SSU rRNA gene, and 28S large subunit (LSU) rRNA gene have been used as DNA barcodes and for phylogenetic studies in different eukaryote taxonomic groups. Because of their popularity, the National Center for Biotechnology Information (NCBI) receives a disproportionate number of rRNA sequence submissions and BLAST queries. These sequences vary in quality, length, origin (nuclear, mitochondria, plastid), and organism source and can represent any region of the ribosomal cistron.ResultsTo improve the timely verification of quality, origin and loci boundaries, we developed Ribovore, a software package for sequence analysis of rRNA sequences. The ribotyper and ribosensor programs are used to validate incoming sequences of bacterial and archaeal SSU rRNA. The ribodbmaker program is used to create high-quality datasets of rRNAs from different taxonomic groups. Key algorithmic steps include comparing candidate sequences against rRNA sequence profile hidden Markov models (HMMs) and covariance models of rRNA sequence and secondary-structure conservation, as well as other tests. At least nine freely available blastn rRNA databases created and maintained with Ribovore are used either for checking incoming GenBank submissions or by the blastn browser interface at NCBI or both. Since 2018, Ribovore has been used to analyze more than 50 million prokaryotic SSU rRNA sequences submitted to GenBank, and to select at least 10,435 fungal rRNA RefSeq records from type material of 8,350 taxa.ConclusionRibovore combines single-sequence and profile-based methods to improve GenBank processing and analysis of rRNA sequences. It is a standalone, portable, and extensible software package for the alignment, classification and validation of rRNA sequences. Researchers planning on submitting SSU rRNA sequences to GenBank are encouraged to download and use Ribovore to analyze their sequences prior to submission to determine which sequences are likely to be automatically accepted into GenBank.

scholarly journals Detection of Introns in Eukaryotic Small Subunit Ribosomal RNA Gene Sequences

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Dipankar Bachar ◽  
Laure Guillou ◽  
Richard Christen
Keyword(s):  
Ribosomal Rna ◽  
Phylogenetic Analyses ◽  
Small Subunit ◽  
Ssu Rrna ◽  
Gene Sequences ◽  
Gene Encoding ◽  
C Program ◽  
Horizontal Transfers ◽  
Sequence Quality ◽  
Rrna Sequences

The gene encoding SSU-rRNA sequences is the tool of choice for phylogenetic analyses and environmental biodiversity analyses of bacteria, Archaea but also unicellular Eukaryota. In Eukaryota, gene sequences may often be interrupted by long or several introns. Searching in GenBank release 188, we found descriptions of 3638 such sequences. Using a database of 180 000 SSU-rRNA sequences well annotated for taxonomy and a C++ program written for that purpose, we computed the presence of 18 691 introns (among which the 3638 described introns). Filtering on length and sequence quality, 3646 sequences were retained. These introns were clustered; clusters were analyzed for the presence of single or multiple clades at various levels of taxonomic depth, allowing future analyses of horizontal transfers. Various analyses of the results are provided as tabulated files as well as FASTA files of described or computed introns. Each sequence is annotated for cellular location (nuclear, chloroplast, and mitochondria), positions at which they were found in the SSU-rRNA sequences and taxonomy as provided by GenBank.

scholarly journals Three Redescriptions in Tintinnopsis (Protista ：Ciliophora: Tintinnina) from Coastal Waters of China, with Cytology and Phylogenetic Analyses Based on Ribosomal RNA Genes

2020 ◽  
Author(s):  
Yang Bai ◽  
Rui Wang ◽  
Wen Song ◽  
Lifang Li ◽  
Luciana F. Santoferrara ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Coastal Waters ◽  
Phylogenetic Analyses ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Closely Related Species ◽  
Molecular Information ◽  
Ciliary Tuft ◽  
Rna Genes ◽  
Rrna Sequences

Abstract Background: The taxonomy of tintinnine ciliates is vastly unresolved because it has traditionally been based on the lorica (a secreted shell) and it has only recently incorporated cytological and molecular information. Tintinnopsis, the most speciose tintinnine genus, is also the most problematic: it is known to be non-monophyletic, but it cannot be revised until more of its species are studied with modern methods.Results: Here, T. hemispiralis Yin, 1956, T. kiaochowensis Yin, 1956, and T. uruguayensis Balech, 1948, from coastal waters of China, were studied. Lorica and cell features were morphometrically investigated in living and protargol-stained specimens, and sequences of three ribosomal RNA (rRNA) genes were phylogenetically analyzed. The three species show a complex ciliary pattern (with ventral, dorsal, and posterior kineties and right, left, and lateral ciliary fields), but differ in lorica morphology, details of the somatic ciliature and rRNA gene sequences. Tintinnopsis hemispiralis is further distinguished by a ciliary tuft (a ribbon of very long cilia originating from the middle portion of the ventral kinety and extending out of the lorica) and multiple macronuclear nodules. Both T. kiaochowensis and T. uruguayensis have two macronuclear nodules, but differ in the number of somatic kineties and the position of the posterior kinety. Two neotypes are fixed for T. hemispiralis and T. kiaochowensis to stabilize the species names objectively, mainly because of the previous unavailability of type materials. By phylogenetic analysis and comparison with closely-related species, we infer that the ciliary tuft and details such as the commencement of the rightmost kinety in the lateral ciliary field are synapomorphies that may help clarify the systematics of Tintinnopsis-like taxa.Conclusion: The redescriptions of three poorly known Tintinnopsis species, namely T. hemispiralis, T. kiaochowensis, and T. uruguayensis firstly revealed their ciliary pattern and rRNA sequences. This study expands knowledge and database of tintinnines and helps in identifying potential synapomorphies for future taxonomic rearrangements.

scholarly journals Microbiological and Geochemical Heterogeneity in an In Situ Uranium Bioremediation Field Site

2005 ◽  
Vol 71 (10) ◽  
pp. 6308-6318 ◽  
Author(s):  
Helen A. Vrionis ◽  
Robert T. Anderson ◽  
Irene Ortiz-Bernad ◽  
Kathleen R. O'Neill ◽  
Charles T. Resch ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Acid Volatile Sulfide ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Geochemical Heterogeneity

ABSTRACT The geochemistry and microbiology of a uranium-contaminated subsurface environment that had undergone two seasons of acetate addition to stimulate microbial U(VI) reduction was examined. There were distinct horizontal and vertical geochemical gradients that could be attributed in large part to the manner in which acetate was distributed in the aquifer, with more reduction of Fe(III) and sulfate occurring at greater depths and closer to the point of acetate injection. Clone libraries of 16S rRNA genes derived from sediments and groundwater indicated an enrichment of sulfate-reducing bacteria in the order Desulfobacterales in sediment and groundwater samples. These samples were collected nearest the injection gallery where microbially reducible Fe(III) oxides were highly depleted, groundwater sulfate concentrations were low, and increases in acid volatile sulfide were observed in the sediment. Further down-gradient, metal-reducing conditions were present as indicated by intermediate Fe(II)/Fe(total) ratios, lower acid volatile sulfide values, and increased abundance of 16S rRNA gene sequences belonging to the dissimilatory Fe(III)- and U(VI)-reducing family Geobacteraceae. Maximal Fe(III) and U(VI) reduction correlated with maximal recovery of Geobacteraceae 16S rRNA gene sequences in both groundwater and sediment; however, the sites at which these maxima occurred were spatially separated within the aquifer. The substantial microbial and geochemical heterogeneity at this site demonstrates that attempts should be made to deliver acetate in a more uniform manner and that closely spaced sampling intervals, horizontally and vertically, in both sediment and groundwater are necessary in order to obtain a more in-depth understanding of microbial processes and the relative contribution of attached and planktonic populations to in situ uranium bioremediation.

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