|
One of the most rapidly evolving scientific technologies in recent years has been next-generation sequencing, a non-Sanger based method with dramatic improvement in speed, accuracy and costeffectiveness. This high-throughput method has provided unexpected opportunities for application in functional genomic research, including gene expression profiling, small non-coding RNA identification and profiling, epigenetic modifications and genetic profiling of complex diseases such as cancer, diabetes and cardiovascular disease. Its application in the growing field of personalised medicine and pharmacogenomics demonstrates both the possibilities and challenges of next-generation sequencing: data management and analysis of sequencing data to cope with the enormous data flood, and its applicability to analyse larger patient cohorts in reasonable timeframes. The latter requires sophisticated, automated and user-friendly techniques to minimise costs and increase throughput. Continued incorporation of advanced technologies in next-generation sequencing will accelerate its establishment in personalised molecular medicine in the near future.
NEXT-GENERATION SEQUENCING
The completion of the Human Genome Project took more than 10 years, required hundreds of sequencers operated by cohorts of personnel, and amounted to $2.7 billion in overall costs. The completion of the sequence of the human genome was one of the key developments in human genomics over the last decade and was crucial for the study of evolution and diseases, as well as for advancements in pharmacogenomics, ultimately leading to a faster, cheaper and more automated sequencing technology for high-throughput and parallel sequencing: next-generation sequencing. This new technology has completely changed genomic research. An entire human genome can be sequenced within eight weeks for less than $100,000 and several providers have already promised the latest era of genome sequencing technology: the ‘$1,000 genome’.
Two landmark studies have revolutionised the sequencing technology. Instead of clone-based libraries, the new platforms use less work-intensive fragment libraries by annealing platform specific linkers to fragmented DNA directly from the genomic DNA (1,2). These adaptor sequences enable amplifications of the fragments without cloning. Established platforms use agarose beads, oligo-derivatised surfaces of a flow-cell, or magnetic beads to isolate genomic DNA-fragments (3). The original Sanger biochemistry is replaced with alternative strategies. The 454 platform established pyrosequencing, a method to detect nucleotide incorporation via ATP-activated reporter firefly luciferase on a ‘sequencing by synthesis’ principle. The sequencing process of the Solexa platform uses fluorescently labelled reversible dNTP terminators to halt the polymerisation step temporarily and determine the bound fluorescence labels. The third platform, SOLiDTM, sequences by hybridisation and ligation and uses a set of fluorescently labelled hybridisation probes. All these platforms sequence with an accuracy of 99.5 per cent or higher, with run times from 7.5 hours up to seven days (4). |
