DNA sequencing has come a long way since the publication of the first sequencing methodology paper in 1977 by Fred Sanger and Alan Coulson. The next-generation sequencing instruments available today, which are based on non-Sanger sequencing technologies, offer unprecedented speed and cost-effective ways of sequencing large genomes that may have been previously considered impossible. The next next-generation instruments that are currently in development with an eye toward personal genomics are looking to offer even greater throughput and cost-efficiency to enable the resequencing of the human genome at around $1,000!
Most DNA sequencers that are in use today for high-throughput large-scale applications are fully automated but vary considerably in the sequencing technology they use. Capillary electrophoresis– based systems are considered traditional automated technology and can incorporate UV or infrared detection of sequencing products. Infrared detection provides the benefit of lower background noise and increased sensitivity compared to other methods. These systems typically process between 400 and 1,000 bases per run.
Sequencing by ligation represents an alternative strategy that incorporates clonal amplification of the DNA. These systems offer greater accuracy (99.99%) and greater throughput (up to 30 gigabases per run) compared to traditional sequencers using capillary electrophoresis. Sequencing-by-synthesis instruments incorporate single nucleotides to generate either a chemiluminescent reaction recorded by a CCD camera or a fluorescent signal captured by UV detection, resulting in read lengths of 400 to 500 bases per run.
The important factors to be considered in the evaluation of different systems are the throughput of processing samples, the cost associated per sample and the capability of the sequencer to process suitable read lengths at a given accuracy. Run cost remains a significant issue when comparing various instruments and technologies. Some sequencing systems generate more reads but offer much shorter read lengths. Next-generation instruments such as the Applied Biosystem’s SOLiD System, the 454 Genome Sequencer FLX marketed by Roche Applied Science and Illumina’s Genome Analyzer are quite comparable when it comes to cost and efficiency. Although these systems require longer run times (days) than traditional sequencing systems, they permit analysis of significantly larger sample sizes compared to other traditional capillary electrophoresis systems.
Although the most obvious application for a DNA sequencer is to identify genetic sequences, it can also be used for other applications such as the detection of single nucleotide polymorphisms or gene expression profiling. As sequencing technologies have evolved to provide faster and more reliable outputs, the sequencing of wholeorganism genomes has become fairly routine for metagenomic, transcriptomic and other large-scale sequencing applications.
There are also a few things to consider when setting up a DNA sequencing laboratory. Lynn Rasmussen, who is currently at the High-Throughput Screening (HTS) Center at the Southern Research Institute, was formerly the manager of a DNA sequencing core lab at the NCI Frederick Cancer Research Center. According to her, setting up a sequencing lab is quite different from setting up an HTS lab. “In a DNA sequencing lab, the samples change but the methodology and reagents that you use to sequence change very rarely,” says Rasmussen. “So once you have the automation set up, programmed and working correctly, you rarely have to make any changes to accommodate new processes.” However, in an HTS lab every assay is different and each one has some nuances that need to be addressed. Very often in a sequencing laboratory certain instruments have to be dedicated to certain use, to avoid DNA contamination across samples and across various runs. “For instance, you may want your equipment for PCR separate from that for DNA sequencing cleanup. You always want to be running these different samples on different equipment.”
Although the debate between using fixed versus disposable pipette tips continues, Rasmussen recommends using disposable tips, especially for liquid handlers in a sequencing lab, where cross-contamination can be a big problem. “If pipette-tip cost is of great concern, then the better approach would be to use a non-contacttype dispenser, which would eliminate the cost of disposables and of any cross-contamination,” she says. “It doesn’t take you many years to recover the additional costs of buying the non-contacttype equipment when you factor in the cost of consumables.”
Tanuja Koppal, PhD, is a freelance science writer and consultant based in Randolph, N.J.
The Agencourt Genomic Services group has purchased an Illumina Genome Analyzer II (GAII) for its next-generation sequencing suite. The comprehensive line-up of sequencing instruments allows Agencourt to address a range of sample and project types for both pharmaceutical and academic researchers. Low library construction input requirements and a variety of kitted applications make the Illumina platform well-suited for many research projects. The company recently upgraded to version 3 SOLiD instruments, and its experience with that platform surpasses that of any sequence provider. The long reads provided by the 454 Life Sciences GS FLX systems and Titanium reagents have utility in a variety of scientific applications and are particularly valuable for de novo sequencing projects.
The SureSelect Target Enrichment system greatly streamlines next generation sequencing by letting scientists sequence only genomic areas of interest. It is a ready-to-use kit containing customer-specified mixtures of up to 55,000 RNA probes in a single tube. Probes are 120 mer, the longest available for this application, making them very effective at capturing unknown mutations. Kits are packaged for studies ranging from tens to thousands of samples, and are well-suited for automation in very high-throughput workflows. SureSelect is available for the Illumina Genome Analyzer and is being optimized for the SOLiD System.
Beckman Coulter and NuGEN Technologies have introduced an automated workstation that allows researchers starting with formalin-fixed, paraffin-embedded (FFPE) tissue samples to produce labeled cDNA fragments ready for gene expression analysis. The verified solution consists of NuGEN Ovation ® gene amplification and labeling systems and the Agencourt® FormaPure® system for isolating total nucleic acids from FFPE samples, in a method implemented on the Beckman Coulter Biomek® FXp Laboratory Automation Workstation. Using the integrated system, researchers will be able to isolate, purify, amplify and label the typically degraded RNA in FFPE samples in a flexible, fully automated process that increases accuracy, frees up valuable time, and delivers biotinylated cDNA fragments ready for global gene expression analysis for biomarker identification using Affymetrix GeneChip arrays.
Integrated DNA Technologies
The Screening Dicer-substrate siRNA (DsiRNA) duplex product is ideal for small scale in vitro applications, providing researchers with a cost effective and quick solution for their RNAi experiments. All duplexes ship within 4 working days and are QC analyzed by ESI mass spectrometry to verify compound identity. Produced on IDT’s proprietary high-throughput synthesis platform, the manufacturing process includes Affinity purification and has been designed to provide a high quality product at a low cost. Available in either tubes or plates, IDT offers a 2 or 10 nanomole scale yields. DsiRNA methods were developed in a collaborative project between IDT and Prof. John Rossi at the Beckman Research Institute of the City of Hope National Medical Center
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