For the past 30 years, the main method for sequencing DNA has been the Sanger sequencing technology. This technology has been shown to be too slow and prohibitively expensive to perform routine genome sequencing.
For the past 30 years, the main method for sequencing DNA has been the Sanger sequencing technology. Despite continued advances such as the introduction of capillary electrophoresis systems, and a continuing decrease in costs, this technology has been shown to be too slow and prohibitively expensive to perform routine genome sequencing .This fact has lead to the development of so called second generation sequencing technologies, having the potential to sequence the human genome for a couple of thousand dollars in the next coming years.
In October 2005, 454 Life Science, a member of the Roche group, was the first company which introduced such a second generation sequencing system into the Life Science market. Several of the recent breakthrough results in genomic research have been achieved using this new technology since then.
Recently Roche Applied Science and 454 Life Science announced the publication of the 100th peer-reviewed study using the Genome Sequencer System, including 12 papers in Nature, 11 papers in Science, 10 papers in Nucleic Acids Research, or four papers in Cell. These first 100 studies span a diverse group of DNA sequencing applications, including de novo sequencing and re-sequencing of whole genomes, metagenomics, RNA analysis, and targeted sequencing of DNA regions of interest.
This article provides a short overview about some selected breakthroughs achieved by using the system.
Resequencing of the human genome, exome or targeted gene regions: The 454 Sequencing technology is the first 2nd generation sequencing technology used to decipher the genome of an individual human being – the genome of Dr James D Watson. The data are currently been analysed by the Human Genome Sequencing Center at the Baylor College of Medcine, Houston, USA, and are expected to provide many new insights into human genetics.
Another breakthrough in human genome resequencing also has been achieved by the Human Genome Sequencing Center at Baylor College, in cooperation with Roche Nimblegen1. Based on the Roche Nimblegen capturing and enrichment procedure, 6726 unique ‘exon’ segments of approximately 500 bases, and ‘locus-specific’ regions ranging in size from 200kb to 5Mb, were extracted from the human genome and sequenced straight forward on a 454 Life Sciences FLX sequencer. This direct enrichment method avoids the need for the tedious and expensive generation of thousands of small and long range PCR products. Hence, the combination of Nimblegen enrichment and 454 Sequencing is a significant technological milestone on the road to routine sequencing of human genomes.
Analysis of structural variations in the human genome: Researchers at Yale University used 454’s new 100nt long paired end protocol to determine structural differences between human genomes. Comparing just two human genomes, researchers identified more than 1000 different structural variations, suggesting that structural variation is responsible for a larger number of differences between the genomes of two individuals than the more heavily studied single nucleotide polymorhisms2.
Studying the molecular basis for eusociality using expression profiling: Researchers from the University of Illinois in the US used the Genome Sequencer FLX to study the molecular basis of eusociality in wasp colonies. 454 Sequencing enabled the researchers to analyse differences in the gene expression profiles in the brain of wasp queens, gynes, workers, and foundresses3. For the first time it could be shown that the wasp brain gene expression in workers was more similar to foundresses, which show maternal care, than to queens and gynes, which do not. Insulin-related genes were among the differentially regulated genes, suggesting that the evolution of eusociality involved major nutritional and reproductive pathways.
Metagenomics and microbial diversity: Mitchell Sogin and his team at the Marine Biological Laboratory analysed the microbial diversity of deep see samples using the amplicon sequencing procedure of the Genome Sequencer System4. They found that the microbial diversity in their samples was 1-2 orders of magnitude more complex than ever published.
A team from Washington University used 454 Sequencing to identify that the microflora in obese individuals differs significantly from that of lean individuals5. This research led to a startling discovery that injecting bacteria from the guts of obese mice into the guts of a germ-free, lean mice resulted in symptoms of obesity.
In a breakthrough study from Columbia University, researchers used the 454 sequencing technology for a transcriptome-metagenomics approach to identify a significant connection between an RNA virus and the mysterious worldwide collapse of honey bee colonies6.
Ancient DNA: Researchers from Pennsylvania State University in the US used the Genome Sequencer System to re-sequence the wholly mammoth genome7. At the MPI in Leipzig, Germany, Svante Päboö and team are sequencing the Neanderthal genome, with the aim to better understand the evolutionary background of variations in the human genome8.
As mentioned above, this was just a selection of many ground breaking results obtained using the Genome Sequencer FLX System. In our continuing effort to enable research that was previously technically or economically impossible, 454 will introduce a powerful new sequencing kit for the current GS FLX system in summer 2008. The new kit will enable increased throughput (>1GB per day) and read length (>400bases) and, most importantly, will lead to considerably decreasing costs per application.”
Dr Marcus Droege, Global Marketing Director for Genome Sequencing, Roche Applied Science, Penzberg, Germany. www.roche-applied-science.com
References:
- Albert et al. Nature Methods, 2007;
- Korbel, J O et al, Science 27, 2007;
- Toth, A L et al Science 27, 2007;
- Sogin, M L et al, Proc. Natl Acad Sci USA 103 (32), 12115-12120, 2006;
- Turnbaugh, P J et al Nature 444 (7122), 1027-1031, 2006;
- Cox-Foster, D L et al Science 6, 2007;
- Poinar, H N, et al. Science 311 (5759), 392-394,2006;
- Green, R E et al. Nature, 444 (7117), 330-336, 2006.
