Abstract
Comparative analysis of small-subunit ribosomal RNA (ss-rRNA) gene sequences forms the basis for much of what we know about the phylogenetic diversity of both cultured and uncultured microorganisms. As sequencing costs continue to decline and throughput increases, sequences of ss-rRNA genes are being obtained at an ever-increasing rate. This increasing flow of data has opened many new windows into microbial diversity and evolution, and at the same time has created significant methodological challenges. Those processes which commonly require time-consuming human intervention, such as the preparation of multiple sequence alignments, simply cannot keep up with the flood of incoming data. Fully automated methods of analysis are needed. Notably, existing automated methods avoid one or more steps that, though computationally costly or difficult, we consider to be important. In particular, we regard both the building of multiple sequence alignments and the performance of high quality phylogenetic analysis to be necessary. We describe here our fully-automated ss-rRNA taxonomy and alignment pipeline (STAP). It generates both high-quality multiple sequence alignments and phylogenetic trees, and thus can be used for multiple purposes including phylogenetically-based taxonomic assignments and analysis of species diversity in environmental samples. The pipeline combines publicly-available packages (PHYML, BLASTN and CLUSTALW) with our automatic alignment, masking, and tree-parsing programs. Most importantly, this automated process yields results comparable to those achievable by manual analysis, yet offers speed and capacity that are unattainable by manual efforts.
Highlights
Introduction ssRNA gene sequence analysis as a tool for microbial systematics and ecologyPhylogenetic analysis of rRNA gene sequences has led to important advances in microbiology, such as the discovery of a third branch on the tree of life [1] and the realization that the microbes that can be grown in pure culture represent but a small fraction, in terms of both phylogenetic types and total numbers of cells of the microbes, found in nature [2]
The small-subunit ribosomal RNA (ss-rRNA) gene has become a key target for environmental microbiology studies largely because through the use of broadly targeted primers, one can use polymerase chain reaction (PCR) to amplify in a single reaction the ss-rRNA genes from a wide diversity of organisms present in an environmental sample [7,8]
In this paper we describe the ss-rRNA taxonomy and alignment pipeline (STAP) software, its testing, and provide examples of some of the ways it can be used in ss-rRNA analyses
Summary
Introduction ssRNA gene sequence analysis as a tool for microbial systematics and ecologyPhylogenetic analysis of rRNA gene sequences ( ssrRNA, i.e., the small subunit rRNA) has led to important advances in microbiology, such as the discovery of a third branch on the tree of life (the archaea) [1] and the realization that the microbes that can be grown in pure culture represent but a small fraction, in terms of both phylogenetic types and total numbers of cells of the microbes, found in nature [2]. The sequencing of ss-rRNA genes from new species is greatly facilitated by the presence of highly conserved regions at several positions along the gene [4]. The conservation of these regions allows one to design and use broadly targeted oligonucleotide primers that work on a wide diversity of species for both sequencing and amplification by the polymerase chain reaction (PCR). The ss-rRNA gene has become a key target for environmental microbiology studies largely because through the use of broadly targeted primers, one can use PCR to amplify in a single reaction the ss-rRNA genes from a wide diversity of organisms present in an environmental sample [7,8]. As sequencing continues to decrease in cost and difficulty, we believe it will become the preferred option and we focus on sequence analysis here
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
