Is this the answer to fast, cheap DNA sequencing

The consequent demand for faster, cheaper DNA sequencing methods means the next generation of DNA sequencing technology must reduce costs without sacrificing data quality

SANGER sequencing has become widely used by researchers over the past thirty years, revolutionising genetics and genomics. The method’s high accuracy and capacity for de novo sequencing, resequencing, genotyping and fragment analysis caused it to become the gold-standard approach, and the technology is now widely accessible to a broad variety of research institutions. During the past decade, hundreds of microbial and a few higher eukaryote genomes  - including human and murine - have been completely sequenced using this tried and tested method. However, sequencing the larger mammalian genomes has so far depended on large research consortiums with multiple machines, and requires significant man-hours to complete each project.

The consequent demand for faster, cheaper DNA sequencing methods is driving the development of next-generation technologies that will allow more sequencing projects to be completed, without compromising the quality of the data. Ideally, any new technology should be able to accommodate future sequencing applications, but still complement traditional sequencing technologies, enabling many more applications than basic sequencing as researchers move from discovery to validation. The expected ultra-high throughput of next-generation sequencing technologies will allow whole genome surveys to be completed, while the fine-mapping abilities of capillary electrophoresis will provide essential detailed information for targeted studies, such as human identification in forensic applications.

One example of the next-generation DNA sequencing platforms is the SOLiD system, recently developed by Applied Biosystems. It can generate up to three gigabases of useable data in a single run by the massively parallel reading of millions of DNA fragments, making it one of the highest throughput systems available, and the high quality data are suitable for diverse applications such as whole genome sequencing, resequencing, genotyping, gene expression, ChIP and small RNA discovery. Unlike polymerase sequencing approaches, the system uses a proprietary technology called stepwise ligation that can analyse billions of templates simultaneously. In the system, DNA fragments are ligated to a universal sequencing primer and amplified onto polystyrene beads using a water-in-oil emulsion PCR technique. The beads are placed onto a random array and universal sequencing primers are added, along with four different fluorescence-labelled oligo probe sets. The primer is reset after each cycle, which removes noise and makes signal-to-noise ratios much higher compared to Sanger sequencing, giving unambiguous results over the whole length of the read sequence. The arrays are read by high-speed automated bead imaging using the system’s four megapixel camera, and the data are stored for later alignment and analysis.

A particularly important feature of this ligation-based technology is its two-base encoding mechanism that interrogates each base twice for errors during sequencing, removing both measurement and system errors and ensuring high accuracy data, which is particularly important for applications such as SNP detection. By using probes that encode for two bases rather than just one, errors can be easily recognised by unexpected signal mismatches relative to reference DNA and easily distinguished from true SNPs, where two colour changes are required to specify the single base change. 

By combining two-base encoding with the system’s mate-pair library construction, other types of genetic variation are also easily detected, including gene copy number variations, single base and larger duplications, inversions, insertions and deletions. The platform’s proven mate-pair analysis provides scientists with a flexible system that can perform a variety of different functions, including gene expression studies for the detection of low-expressed genes, which can go undetected on hybridisation arrays where a prior knowledge is required. In addition to the instrument itself, the system includes a computing cluster and data storage, basic library preparation chemistry and protocols, emulsion PCR kits, sequencing chemistry kits and ancillary equipment to perform the workflow.

Techniques employed by the SOLiD system

The new platform is designed to accommodate future sequencing applications as it can be scaled through adaptable bead enrichment to support a higher density of sequence per slide – with future plans to extend beyond ten gigabases of useable data per run – providing the infrastructure for performing more complex genomic studies.

Currently, it supports resequencing of bacterial and microbial genomes, and bacterial artificial chromosome (BAC) contigs, which are frequently used for amplifying a short piece of an organism’s DNA that is inserted into the BAC prior to sequencing. A number of software tools and validated methods and protocols are available with the platform for these applications, supporting advances in environmental and viral research, pathogen detection and infectious diseases.

The company say the high sensitivity and accuracy of the technology make it particularly useful for identifying rare genetic mutations in cancer, helping researchers to understand the genetic basis of such complex diseases. It is also expected to be of substantial benefit to pharmaceutical companies searching for suitable drugs, as the high throughput resequencing technology can be used to determine association of specific gene expression patterns with possible toxic drug effects, so helping researchers to identify failed candidate drugs at early stages in the development process. An increasing number of pharmaceutical laboratories are also using genomics technologies to understand the mode of action of drugs for toxicology studies.

Next-generation sequencing technology has the potential to dramatically reduce the cost of DNA sequencing without sacrificing quality of data; to broaden the applications of genomic information in medical research and healthcare, and enable new discoveries that may revolutionise medicine. Sequencing-based expression studies using the SOLiD technology can be used for both discovery and screening applications providing more complete information over other commercially available hybridisation microarrays because it does not require prior knowledge of gene sequences. There is much untapped potential in the new generation sequencing technology that will be extremely useful for expansion of applications in many academic, pharmaceutical and genomics institutions focused on research in the fields of life sciences, agriculture, biofuel production, environmental research, bacteriology and virology.

By Sue Ann Molero. Sue is genetic analysis marketing manager, Europe, for Applied Biosystems.