Transfection of cells is one of the main techniques used to influence gene expression. Most primary cells and human skeletal myoblasts (SkMC) in particular are very difficult to transfect, whereas for cell lines such as C2C12, many suitable transfect more
Metagenomics Analysis Using the Genome Sequencer FLX SystemTom Jarvie1 and Tim Harkins2
1 454 Life Sciences, Branford, USA; 2Roche Applied Science, Indianapolis, USA
The Genome Sequencer FLX System from 454 Life Sciences and Roche Applied Science is a versatile sequencing platform suitable for a wide range of applications, including de novo sequencing and assembly of genomic DNA, transcriptome sequencing, small RNA analysis, and amplicon sequencing. The Genome Sequencer FLX is built upon 454 Sequencing technology that enables long read lengths and very high single-read accuracy. One application in which the technology is accelerating the field’s understanding is metagenomics.
Metagenomics is the study of genomic content in a complex mixture of microorganisms (see Table 1 for other definitions). The two primary goals of this approach are to develop a consensus of what populations of microorganisms are present (a horizontal screen) and then to identify what roles each microorganism has within a specific environment (a vertical characterization). Metagenomics samples are found nearly everywhere, including several micro-environments within the human body, soil samples, extreme environments such as deep mines, and the various layers within the ocean. Therefore, the diversity of microorganisms is thought to be in the range of tens of millions to greater than hundreds of millions of species. The ocean alone is estimated to have 3.6 x 1029 microbial cells. When one considers that microbial organisms have had over 3.5 billion years to mutate, and that horizontal gene transfer is common, it is easy to understand why a vast number of species is predicted.
Recent Publications Have Shown the Vast Diversity of the Microbial World
In one example, two deep mine samples were found to comprise very different communities exhibiting different metabolisms, even though the samples were in very close physical proximity to one another . Interestingly, the microbes studied were completely different from other previously sequenced microbial communities. A second publication, addressing microbial diversity within the ocean, described a screening approach using sequence tags for rRNA . In this study, it was estimated that the microbial diversity was up to three orders of magnitude greater than the previous estimate of 106 species.
Although the roles of most microorganisms have yet to be discovered, several recent publications have shown that microorganisms have both competing and synergistic interactions with each other, and these interactions can change as their local environments change. For example, a study of the human gut found that there are two principal populations of bacteria, the Bacteroidetes and the Firmicutes, and their relative abundance to one another changes as the body fat of the individual changes . As body fat increases, the abundance of Firmicutes increases and this in turn increases the capacity for energy harvest, leading to higher obesity rates.
Advantages of Genome Sequencer Technology
The Genome Sequencer FLX System (Figure 1) offers several advantages for metagenomics studies. The first breakthrough is that it eliminates the requirement to clone DNA fragments into bacteria. Consequently, the Genome Sequencer FLX avoids the cloning bias that is introduced in sample preparation as observed in Sanger sequencing-based methods.
With over 400,000 sequencing reads per instrument run, this system can facilitate extensive surveys to identify large numbers of different genes, metabolic pathways, and microbial species that may be present, while providing a dramatic reduction in the cost per project. This allows researchers to approach samples and address questions that, until recently, only major genome centers could achieve. As a result, the Genome Sequencer FLX System offers a powerful technology that supports research studies to answer environmental and ecological questions.
Sequencing read length is also an important factor for metagenomics studies. As most genomes in metagenomics samples are unknown, and relatively little reference sequence information is available, it is important to have read lengths that allow researchers to both assemble genomes in a de novo fashion and uniquely assign them to a specific gene and/or genome. Other next-generation sequencing technologies use sequencing read lengths, termed “microreads”, which are in the range of 15–40 bp in length. Microreads are limiting in metagenomics due to the homology and repetitive regions within one genome and across the multiple genomes within the sample. The ability to uniquely assign a read to one genome becomes more challenging when there are numerous genomes present within a sample. In addition, microreads prevent the assembly of many of the reads. As previously published, sequencing read lengths of 100 bases are near the minimum from a utility perspective [1,4]. The Genome Sequencer FLX generates sequencing read lengths between 200 to 300 bases in length, where read length is dependent upon the specific sequence characteristics.
Bioinformatics for Metagenomics
The Genome Sequencer FLX generates over 100 million bases per instrument run. For metagenomics studies this presents a very large dataset with several challenges that need to be addressed. To help understand the available bioinformatics resources, we have listed several publicly available websites for additional reference input (Table 2). Typically, the first objective of the analysis is to identify which sequencing reads can be associated with known genomes versus unknown organisms. Using an application such as MEGAN, researchers can group their data based upon a taxonomical level to summarize and order their results. With this approach, researchers can assess the complexity of their samples as to the diversity of microorganisms that are present. Additionally, a 16S rDNA analysis can be performed, which will identify a low number of sequencing reads that can help elucidate which genus or species are present within the sample.
The next objective for many metagenomics studies is to identify the metabolic functions of the microorganism within the sample. With average Genome Sequencer FLX read lengths of 250 bases, it is possible to search against sequence databases for homologs; however, the hit rate will most likely be low, in the 5–10% range (due to the incomplete nature of the microbial sequence databases). However, this will still provide up to 20,000–40,000 sequences to examine for known functionality. By using BLASTX, a CPU-intensive application, a functional analysis can be performed to help identify the metabolic function of the organisms within the metagenomic sample.
The ultimate goal of metagenomics is sequence assemblies resulting in, at a minimum, full-length genes and preferably, complete genomes. The 250-base-pair read lengths generated by the Genome Sequencer FLX allow de novo assemblies of metagenomes. Generating assemblies of the sequencing reads is critical for characterizing full-length genes, discovering new genes, and ultimately to help model microbial communities based upon sequence similarities.
Until recently, metagenomics analysis was limited by the cost, low throughput, and inherent cloning bias of the Sanger technologies. The Genome Sequencer FLX System provides a comprehensive view of metagenomics samples with high throughput, no cloning bias, and read lengths long enough to allow diversity and functional analysis of microbial communities.
Additional information about the Genome Sequencer FLX System is available from Roche Applied Science (www.genome-sequencing.com).
1. Edwards RA et al., 2006, BMC Genomics 7:57
2. Sogin M. et al., 2006, Proc Natl Acad Sci USA 103:12115–12120
3. Turnbaugh PJ et al., 2006, Nature 444:1027–1031
4. Goldberg SM et al., 2006, Proc Natl Acad Sci USA 103:11240–11245
5. Sambrook J, Fritsch EF, and Maniatis T: Molecular Cloning: A Laboratory Manual, 2.74 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).
6. Ausubel FM et al. Short Protocols in Molecular Biology [5th Ed.], Vol. 1:2–11 (John Wiley and Sons, Inc., 2002).
License disclaimer information is available online (www.genome-sequencing.com).
This article was originally published in Biochemica 3/2007, pages 4-6. ©Springer Medizin Verlag 2007
- genome sequencers
- genome sequences
- microbial communities
- small RNA
- sample preparation
- molecular biology
- Life Sciences
- horizontal gene transfer
- genome sequencing
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