Soil Bacterial Diversity Screening Using Single 16S rRNA Gene V Regions Coupled with Multi-Million Read Generating Sequencing Technologies

The novel multi-million read generating sequencing technologies are very promising for resolving the immense soil 16S rRNA gene bacterial diversity. Yet they have a limited maximum sequence length screening ability, restricting studies in screening DNA stretches of single 16S rRNA gene hypervariable (V) regions. The aim of the present study was to assess the effects of properties of four consecutive V regions (V3-6) on commonly applied analytical methodologies in bacterial ecology studies. Using an in silico approach, the performance of each V region was compared with the complete 16S rRNA gene stretch. We assessed related properties of the soil derived bacterial sequence collection of the Ribosomal Database Project (RDP) database and concomitantly performed simulations based on published datasets. Results indicate that overall the most prominent V region for soil bacterial diversity studies was V3, even though it was outperformed in some of the tests. Despite its high performance during most tests, V4 was less conserved along flanking sites, thus reducing its ability for bacterial diversity coverage. V5 performed well in the non-redundant RDP database based analysis. However V5 did not resemble the full-length 16S rRNA gene sequence results as well as V3 and V4 did when the natural sequence frequency and occurrence approximation was considered in the virtual experiment. Although, the highly conserved flanking sequence regions of V6 provide the ability to amplify partial 16S rRNA gene sequences from very diverse owners, it was demonstrated that V6 was the least informative compared to the rest examined V regions. Our results indicate that environment specific database exploration and theoretical assessment of the experimental approach are strongly suggested in 16S rRNA gene based bacterial diversity studies.

[1]  W. D. de Vos,et al.  Comparative Analysis of Pyrosequencing and a Phylogenetic Microarray for Exploring Microbial Community Structures in the Human Distal Intestine , 2009, PloS one.

[2]  Xiao-Tao Jiang,et al.  Effects of polymerase, template dilution and cycle number on PCR based 16 S rRNA diversity analysis using the deep sequencing method , 2010, BMC Microbiology.

[3]  P. Qian,et al.  Conservative Fragments in Bacterial 16S rRNA Genes and Primer Design for 16S Ribosomal DNA Amplicons in Metagenomic Studies , 2009, PloS one.

[4]  Jo Handelsman,et al.  Toward a Census of Bacteria in Soil , 2006, PLoS Comput. Biol..

[5]  R Core Team,et al.  R: A language and environment for statistical computing. , 2014 .

[6]  Leo S. D. Caves,et al.  Bio3d: An R Package , 2022 .

[7]  Susan M. Huse,et al.  Exploring Microbial Diversity and Taxonomy Using SSU rRNA Hypervariable Tag Sequencing , 2008, PLoS genetics.

[8]  P. Schloss A High-Throughput DNA Sequence Aligner for Microbial Ecology Studies , 2009, PloS one.

[9]  Martin Hartmann,et al.  Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities , 2009, Applied and Environmental Microbiology.

[10]  James A. Foster,et al.  Phylogenetics Clearcut : a fast implementation of relaxed neighbor joining , 2006 .

[11]  H. Ochman,et al.  Illumina-based analysis of microbial community diversity , 2011, The ISME Journal.

[12]  James R. Cole,et al.  The Ribosomal Database Project: improved alignments and new tools for rRNA analysis , 2008, Nucleic Acids Res..

[13]  J. Prosser Replicate or lie. , 2010, Environmental microbiology.

[14]  R. Knight,et al.  Quantitative and Qualitative β Diversity Measures Lead to Different Insights into Factors That Structure Microbial Communities , 2007, Applied and Environmental Microbiology.

[15]  J. Handelsman,et al.  Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products. , 1998, Chemistry & biology.

[16]  R. Knight,et al.  UniFrac: a New Phylogenetic Method for Comparing Microbial Communities , 2005, Applied and Environmental Microbiology.

[17]  W. Ludwig,et al.  SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB , 2007, Nucleic acids research.

[18]  Nadine Herold,et al.  Horizon-Specific Bacterial Community Composition of German Grassland Soils, as Revealed by Pyrosequencing-Based Analysis of 16S rRNA Genes , 2010, Applied and Environmental Microbiology.

[19]  D. Cowan,et al.  Review and re-analysis of domain-specific 16S primers. , 2003, Journal of microbiological methods.

[20]  A. Uitterlinden,et al.  Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA , 1993, Applied and environmental microbiology.

[21]  Andrea K. Bartram,et al.  Generation of Multimillion-Sequence 16S rRNA Gene Libraries from Complex Microbial Communities by Assembling Paired-End Illumina Reads , 2011, Applied and Environmental Microbiology.

[22]  J. Tiedje,et al.  Naïve Bayesian Classifier for Rapid Assignment of rRNA Sequences into the New Bacterial Taxonomy , 2007, Applied and Environmental Microbiology.

[23]  G. Bronner,et al.  Comparison of 16S rRNA and protein-coding genes as molecular markers for assessing microbial diversity (Bacteria and Archaea) in ecosystems. , 2011, FEMS microbiology ecology.

[24]  G. Casella,et al.  Pyrosequencing enumerates and contrasts soil microbial diversity , 2007, The ISME Journal.

[25]  I. Schöning,et al.  Pyrosequencing-Based Assessment of Bacterial Community Structure Along Different Management Types in German Forest and Grassland Soils , 2011, PloS one.

[26]  L. Øvreås,et al.  Microbial diversity and function in soil: from genes to ecosystems. , 2002, Current opinion in microbiology.

[27]  K. Hornik,et al.  A Lego System for Conditional Inference , 2006 .

[28]  K. Schleifer,et al.  Phylogenetic identification and in situ detection of individual microbial cells without cultivation. , 1995, Microbiological reviews.

[29]  C. Woese,et al.  Bacterial evolution , 1987, Microbiological reviews.

[30]  Philip Hugenholtz,et al.  NAST: a multiple sequence alignment server for comparative analysis of 16S rRNA genes , 2006, Nucleic Acids Res..

[31]  Hans H. Cheng,et al.  Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA , 1997, Applied and environmental microbiology.

[32]  Matthias Meyer,et al.  Illumina sequencing library preparation for highly multiplexed target capture and sequencing. , 2010, Cold Spring Harbor protocols.

[33]  P. Sassone-Corsi,et al.  Computational Improvements Reveal Great Bacterial Diversity and High Metal Toxicity in Soil , 2022 .

[34]  S. Tringe,et al.  Comparative Metagenomics of Microbial Communities , 2004, Science.