In silico analysis of the 16S rRNA gene of endophytic bacteria, isolated from the aerial parts and seeds of important agricultural crops

ABSTRACT Phylogenetic and “fingerprinting” analyses of the 16S rRNA genes of prokaryotes have been a mainstay of microbial ecology during the last two decades. However, many methods and results from studies that rely on the 16S rRNA gene for detection and quantification of specific microbial taxa have seemingly received only cursory or even no validation. To directly examine the efficacy and specificity of 16S rRNA gene-based primers for phylum-, class-, and operational taxonomic unit-specific target amplification in quantitative PCR, we created a collection of primers based solely on an extensive soil bacterial 16S rRNA gene clone library containing ∼5,000 sequences from a single soil sample (i.e., a closed site-specific library was used to create PCR primers for use at this site). These primers were initially tested in silico prior to empirical testing by PCR amplification of known target sequences and of controls based on disparate phylogenetic groups. Although all primers were highly specific according to the in silico analysis, the empirical analyses clearly exhibited a high degree of nonspecificity for many of the phyla or classes, while other primers proved to be highly specific. These findings suggest that significant care must be taken when interpreting studies whose results were obtained with target specific primers that were not adequately validated, especially where population densities or dynamics have been inferred from the data. Further, we suggest that the reliability of quantification of specific target abundance using 16S rRNA-based quantitative PCR is case specific and must be determined through rigorous empirical testing rather than solely in silico.

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The Impact of Primer Design on Amplicon-Based Metagenomic Profiling Accuracy: Detailed Insights into Bifidobacterial Community Structure

Microorganisms ◽

10.3390/microorganisms8010131 ◽

2020 ◽

Vol 8 (1) ◽

pp. 131 ◽

Cited By ~ 6

Author(s):

Leonardo Mancabelli ◽

Christian Milani ◽

Gabriele Andrea Lugli ◽

Federico Fontana ◽

Francesca Turroni ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

In Silico ◽

In Silico Analysis ◽

Amplification Efficiency ◽

Rrna Gene ◽

Test Case ◽

Bacterial Populations ◽

Taxonomic Marker ◽

The Impact

Next Generation Sequencing (NGS) technologies have overcome the limitations of cultivation-dependent approaches and allowed detailed study of bacterial populations that inhabit the human body. The consortium of bacteria residing in the human intestinal tract, also known as the gut microbiota, impacts several physiological processes important for preservation of the health status of the host. The most widespread microbiota profiling method is based on amplification and sequencing of a variable portion of the 16S rRNA gene as a universal taxonomic marker among members of the Bacteria domain. Despite its popularity and obvious advantages, this 16S rRNA gene-based approach comes with some important limitations. In particular, the choice of the primer pair for amplification plays a major role in defining the accuracy of the reconstructed bacterial profiles. In the current study, we performed an in silico PCR using all currently described 16S rRNA gene-targeting primer pairs (PP) in order to assess their efficiency. Our results show that V3, V4, V5, and V6 were the optimal regions on which to design 16S rRNA metagenomic primers. In detail, PP39 (Probio_Uni/Probio_Rev), PP41 (341F/534R), and PP72 (970F/1050R) were the most suitable primer pairs with an amplification efficiency of >98.5%. Furthermore, the Bifidobacterium genus was examined as a test case for accurate evaluation of intra-genus performances at subspecies level. Intriguingly, the in silico analysis revealed that primer pair PP55 (527f/1406r) was unable to amplify the targeted region of any member of this bacterial genus, while several other primer pairs seem to rather inefficiently amplify the target region of the main bifidobacterial taxa. These results highlight that selection of a 16S rRNA gene-based PP should be done with utmost care in order to avoid biases in microbiota profiling results.

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Endophytic bacteria isolated from garden pea (Pisum sativum L.)

Abstract book of the 2nd International Scientific Conference "Plants and Microbes: the Future of Biotechnology" PLAMIC2020 ◽

10.28983/plamic2020.265 ◽

2020 ◽

Author(s):

E. N. Vasileva ◽

A. M. Afonin ◽

G. A. Akhtemova ◽

V. A. Zhukov ◽

I. A. Tikhonovich

Keyword(s):

16S Rrna ◽

Pisum Sativum ◽

16S Rrna Gene ◽

Endophytic Bacteria ◽

Taxonomic Status ◽

Rrna Gene ◽

Garden Pea ◽

Aerial Parts ◽

Pisum Sativum L ◽

Growth Promoting

Endophytic bacteria were isolated from surface-sterilized aerial parts of pea. Taxonomic status of isolated strains was determined by sequencing of 16S rRNA gene. Moreover, genomes of growth-promoting endophytes were sequenced.

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‘Candidatus Phytoplasma stylosanthis’, a novel taxon with a diverse host range in Australia, characterised using multilocus sequence analysis of 16S rRNA, secA, tuf, and rp genes

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijsem.0.004589 ◽

2020 ◽

Author(s):

Bianca Rodrigues Jardim ◽

Wycliff M. Kinoti ◽

Lucy T. T. Tran-Nguyen ◽

Cherie Gambley ◽

Brendan Rodoni ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

In Silico ◽

Phylogenetic Trees ◽

Gene Tree ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Pattern Similarity ◽

The 16S Rrna Gene

In Australia, Stylosanthes little leaf (StLL) phytoplasma has been detected in Stylosanthes scabra Vogel, Arachis pintoi Krapov, Saccharum officinarum L., Carica papaya L., Medicago sativa L., and Solanum tuberosum L. The 16S rRNA gene sequence of StLL phytoplasma strains from S. scabra, C. papaya, S. officinarum and S. tuberosum were compared and share 99.93–100 % nucleotide sequence identity. Phylogenetic comparisons between the 16S rRNA genes of StLL phytoplasma and other ‘Candidatus Phytoplasma’ species indicate that StLL represents a distinct phytoplasma lineage. It shares its most recent known ancestry with ‘Ca. Phytoplasma luffae’ (16SrVIII-A), with which it has 97.17–97.25 % nucleotide identity. In silico RFLP analysis of the 16S rRNA amplicon using iPhyClassifier indicate that StLL phytoplasmas have a unique pattern (similarity coefficient below 0.85) that is most similar to that of ‘Ca. Phytoplasma luffae’. The unique in silico RFLP patterns were confirmed in vitro. Nucleotide sequences of genes that are more variable than the 16S rRNA gene, namely tuf (tu-elongation factor), secA (partial translocation gene), and the partial ribosomal protein (rp) gene operon (rps19-rpl22-rps3), produced phylogenetic trees with similar branching patterns to the 16S rRNA gene tree. Sequence comparisons between the StLL 16S rRNA spacer region confirmed previous reports of rrn interoperon sequence heterogeneity for StLL, where the spacer region of rrnB encodes a complete tRNA-Isoleucine gene and the rrnA spacer region does not. Together these results suggest that the Australian phytoplasma, StLL, is unique according to the International Organization for Mycoplasmology (IRPCM) recommendations. The novel taxon ‘Ca. Phytoplasma stylosanthis’ is proposed, with the most recent strain from a potato crop in Victoria, Australia, serving as the reference strain (deposited in the Victorian Plant Pathology Herbarium as VPRI 43683).

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In silico Approach to Study Adaptive Divergence in Nucleotide Composition of the 16S rRNA Gene Among Bacteria Thriving Under Different Temperature Regimes

Journal of Computational Biology ◽

10.1089/cmb.2014.0116 ◽

2014 ◽

Vol 21 (10) ◽

pp. 753-759 ◽

Cited By ~ 3

Author(s):

Hari Ram ◽

Alok Kumar ◽

Lebin Thomas ◽

Ved Pal Singh

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

In Silico ◽

Nucleotide Composition ◽

Adaptive Divergence ◽

Rrna Gene ◽

Temperature Regimes ◽

The 16S Rrna Gene

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Evaluation of Compatibility of 16S rRNA V3V4 and V4 Amplicon Libraries for Clinical Microbiome Profiling

10.1101/2020.08.18.256818 ◽

2020 ◽

Author(s):

Po-Yu Liu ◽

Wei-Kai Wu ◽

Chieh-Chang Chen ◽

Suraphan Panyod ◽

Lee-Yan Sheen ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

In Silico ◽

Rrna Gene ◽

Systematic Bias ◽

Fecal Microbiome ◽

Hypervariable Regions ◽

Microbiome Profiling ◽

The 16S Rrna Gene ◽

Generation Sequencing

ABSTRACTSequencing of the 16S rRNA gene by Illumina next-generation sequencing is broadly used in microbiome studies. Different hypervariable regions of the 16S rRNA gene, V3V4 (amplified with primers 341F–805R) or V4 (V4O; primers 515F–806R), are selected, depending on the targeted resolution. However, in population-based clinical studies, combining V3V4 and V4 data from different studies for a meta-analysis is challenging. Reads generated by short-read (150-bp) high-throughput sequencing platforms do not fully recover the V4 region read-length. Here, we evaluated the compatibility of 16S rRNA V3V4 and V4 amplicons for microbiome profiling. We compared taxonomic compositions obtained by the analysis of V3V4 and V4 amplicons, and V4 fragments trimmed from V3V4 amplicons. We also evaluated an alternative V4 region (V4N; primers 519F–798R) designed for efficient stitching with 150-bp paired-end sequencing. First, we simulated a global investigation of environmental prokaryotes in silico. This revealed that V4O primers recovered the highest proportion of fragments (81.7%) and most phyla, including archaea. Empirical sequencing of standard (mock) and human fecal samples revealed biased patterns of each primer that were similar to the ones determined by in silico simulation. Further, for human fecal microbiome profiling, the between-sample variance was greater than the systematic bias of each primer. The use of trimmed V4 fragments and single-end amplicons resulted in the same systematic bias. In conclusion, paired-end V4O sequencing yielded the most accurate data for both, simulation and mock community sequencing; the V4O amplicons were compatible with trimmed V4 sequences for microbiome profiling.IMPORTANCENext-generation sequencing of the 16S rRNA gene is a commonly used approach for clinical microbiome studies. Different amplicons of the 16S rRNA hypervariable regions are used in different studies, which creates incompatible sequence features when comparing and integrating data among studies by using 16S denoising pipelines. Here we compared the type of data and coverage obtained when different 16S rRNA amplicons were analyzed. In silico and empirical analyses of the human fecal microbiome revealed that the V3V4 amplicons are compatible with V4 amplicons after trimming up to the same region. These observations demonstrate that reconciling the compatibility of clinical microbiome data from different studies improve not only the sample size but also the confidence of the hypothesis tested.

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In silico analysis of 16S rRNA gene sequencing based methods for identification of medically important aerobic Gram-negative bacteria

Journal of Medical Microbiology ◽

10.1099/jmm.0.027805-0 ◽

2011 ◽

Vol 60 (9) ◽

pp. 1281-1286 ◽

Cited By ~ 9

Author(s):

Jade L. L. Teng ◽

Ming-Yiu Yeung ◽

Geoffrey Yue ◽

Rex K. H. Au-Yeung ◽

Eugene Y. H. Yeung ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Gene Sequencing ◽

In Silico Analysis ◽

16S Rrna Gene Sequencing ◽

Rrna Gene ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Rrna Gene Sequencing ◽

Silico Analysis

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Phyllobacterium loti sp. nov. isolated from nodules of Lotus corniculatus

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.052993-0 ◽

2014 ◽

Vol 64 (Pt_3) ◽

pp. 781-786 ◽

Cited By ~ 33

Author(s):

Maximo Sánchez ◽

Martha-Helena Ramírez-Bahena ◽

Alvaro Peix ◽

María J. Lorite ◽

Juan Sanjuán ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Sequence Similarity ◽

Carbon Sources ◽

Lotus Corniculatus ◽

Culture Conditions ◽

Rrna Gene ◽

Content Type ◽

Link Type ◽

The 16S Rrna Gene

Strain S658T was isolated from a Lotus corniculatus nodule in a soil sample obtained in Uruguay. Phylogenetic analysis of the 16S rRNA gene and atpD gene showed that this strain clustered within the genus Phyllobacterium . The closest related species was, in both cases, Phyllobacterium trifolii PETP02T with 99.8 % sequence similarity in the 16S rRNA gene and 96.1 % in the atpD gene. The 16S rRNA gene contains an insert at the beginning of the sequence that has no similarities with other inserts present in the same gene in described rhizobial species. Ubiquinone Q-10 was the only quinone detected. Strain S658T differed from its closest relatives through its growth in diverse culture conditions and in the assimilation of several carbon sources. It was not able to reproduce nodules in Lotus corniculatus. The results of DNA–DNA hybridization, phenotypic tests and fatty acid analyses confirmed that this strain should be classified as a representative of a novel species of the genus Phyllobacterium , for which the name Phyllobacterium loti sp. nov. is proposed. The type strain is S658T( = LMG 27289T = CECT 8230T).

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