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Study diversity in environmental microbial communities using the GS FLX and GS Junior Systems. 454 Sequencing Systems have become the standard for ribosomal RNA identification (i.e. 16S, 18S, etc.) and whole genome surveys having enabled an unprecedented view of microbial diversity in such environments as the human gut and mouth, soil, coral reefs, deep sea thermal vents, drinking water, and much more. The clonal reads provided by 454 Sequencing Systems enable population characterization without laborious cloning protocols.

16S and 18S ribosomal RNA (rRNA) – Long reads span multiple variable regions, enabling high-resolution species identification (Figure).

Shotgun survey – Long reads enable more unique alignment when searching reference databases, cover longer portions of genes for novel gene discovery, and enable de novo assemblies.

figure 3. Illustration of the 16S rRNA subunit showing possible candidate variable region coverage using long GS FLX Titanium or GS Junior Titanium amplicons. Using birdirectional amplicons, any two (and sometimes three) contiguous variable regions can be covered, while unidirectional amplicons can cover two, three, or even four contiguous variable regions, for greater specificity in phylogenic grouping.

16S and 18S rRNA Amplicon Sequencing

In this approach, rRNA gene PCR products (amplicons) are designed to cover predetermined variable regions of the 16S and/or of the 18S rRNA gene. Organism-specific differences in the sequence of variable regions allow identification of the source organism for individual reads using a BLAST search or other mapping strategies. Long 454 Sequencing reads cover multiple variable regions in a single read, offering higher levels of phylogenetic identification and increased confidence in taxonomic assignment.

The merits of sequencing the different variable regions of rRNA genes are discussed in a variety of publications. Wu GD et al. discuss the performance of different 16S amplicons in the publication Sampling and pyrosequencing methods for characterizing bacterial communities in the human gut using 16S sequence tags. Wu GD et al. (2010) BMC Microbiology 10(1): 206.

Shotgun Metagenomics

Shotgun metagenomics is a random fragment sequencing application that is used on a sample derived from a pool of organisms. Long 454 Sequencing reads provide a significant advantage in shotgun metagenomics experiment as they are better able to distinguish between genes from related organisms, thereby providing a more accurate picture of diversity than shorter reads. Long reads also enable de novo assembly of reads within metagenomics samples.

cDNA Metagenomics - Pathogen Detection

Sequencing viral RNA and DNA in a sample has been useful in discovering novel pathogenic organisms. Assembly of long 454 Sequencing reads, resulting in a substantial portion of the viral genome, is often sufficient for identification of novel viruses. Since viral RNA genomes may make up only a small fraction of the total RNA, the source of the sample can determine success or failure. Ideally, the source will contain a significant titer of viral genomes or a reduction in cellular RNA.

Isolating DNA and RNA viruses from samples prior to sequencing focuses the sequencing bandwidth on the viral components. Removal of cellular RNA and DNA is typically accomplished by nuclease treatment while the viruses are intact. After nucleases are inactivated, the viral capsids are dissociated and the viral nucleic acids are purified, subjected to cDNA synthesis, amplification, and 454 Adaptor ligation, which purifies both DNA and RNA genomes. Example protocols can be found in Briese et al. Genetic Detection and Characterization of Lujo Virus, a New Hemorrhagic Fever–Associated Arenavirus from Southern Africa. PLoS Pathogens. (2009), 5(5): e1000455 and in other publications such as Onions and Kolman, Massively parallel sequencing, a new method for detecting adventitious agents. Biologicals. (2010), 38(3): 377-380.

There is a wide variety of publicly available tools for the analyses of metagenomic data. One or more of the public tools described in the list below may be useful, but it is up to the individual researcher to determine which suits their experimental goals best. MEGAN, a Metagenome Analyzer, allows a single scientist to analyze large data sets and group sequencing reads into taxonomic units. MG-RAST is a fully-automated service for annotating metagenome samples. IMG/M provides tools for analyzing the functional capability of microbial communities based on their metagenome sequence, in the context of reference isolate genomes, using a variety of public functional and pathway resources. CAMERA is a user-driven site dedicated to providing the scientific community with metagenomics data and analysis tools. - CARMA is a software pipeline for the characterization of species composition and the genetic potential of microbial samples using unassembled reads. - Galaxy includes higher eukaryotes, such as insects in the analysis pipeline. - Greengenes provides access to comprehensive 16S rRNA gene sequence alignment - QIIME provides a full workflow for processing 454 16S metagenomics experiments. - The ribosomal database project has a pyrosequencing specific pipeline.

The above list is meant to provide examples of the types of analysis that are available, but the rapid evolution in this field necessitates that researchers investigate the options that are best suited for their needs.