Sequencing Facility
* The IGSP DNA Sequencing Facility is a resource managed by Lisa Bukovnik, and is available to the Duke research community. The facility began as a Duke Biology resource lab in 1999 under faculty advisor of Rytas Vilgalys, with one ABI 3700 capillary sequencing instrument. Lisa has been the lab's director since 2000. In 2006, the lab's new faculty advisor, Greg Wray, took the facility into the IGSP as part of the Center for Evolutionary Genomics, where the Sequencing Facility became a campus-wide core service facility.
* The Facility now houses a variety of DNA sequencing instruments and their support equipment. Routine DTR and capillary electrophoresis of customer prepared sequencing and genotyping samples is performed on a daily basis using two Applied Biosystems 3730xl DNA Analyzers. The 3730’s generate 950-base reads for 2000 sequencing samples in 24 hours, or 3200 genotypes. An Applied Biosystems 7000 Real-Time PCR instrument is maintained by the Facility for Biology account holders. Our next-generation sequencing quiver includes one Roche GS-FLX (454) and three Illumina Genome Analyzer II's.
Roche GS-FLX
Roche GS-FLX (aka “the 454”)In 2008, two ultra-high throughput instruments were installed in the facility: a Roche GS-FLX (aka “the 454”) System and an Illumina Genome Analyzer (GA), both of which are available for campus-wide use. The GS-FLX is considered a “long-read” platform, and can generate 400,000 mappable reads at 250 bases (100 million bases) in one 8-hour run. The 454 is especially good for de novo sequencing, resequencing, transcriptome analysis, gene regulation, epigenetic changes, metagenomics, and microbial diversity. It was the platform used for elucidating the wooly mammoth genome, and is currently is use on the Neanderthal project.
Here at Duke, the full capabilities of the 454 are being explored on the Armaleo/Lutzoni lichen genome project, including the Long Paired End protocol, and the 435-base read “Titatnium” upgrade. Duke's 454 has been used in Duke Pediatrics researcher Patrick Seed's "baby gut microbiome" project, and has been utilized by Duke Biology's 2009 Darwin-Wallace Medal Winner, Mohamed Noor. We are looking forward to assisting Duke's Erich Jarvis with his Parrot Genome Project in the spring of 2009.
To plan your own 454 project, go to Steps to Running Your 454 Project.
Illumina GAII
The Illumina GAII (aka “Solexa”)The Illumina GAII (aka “Solexa”), considered a “short read” platform, can generate 1.5 Gbp in 35-base reads to give 50 million reads per flowcell, in one 2-day run. The short read instruments are invaluable in their power to elucidate gene expression via mRNA tag profiling, small RNA discovery and analysis, whole transcriptome profiling, and medical resequencing. The Sequencing Facility accepted the delivery of a second GAII on September 26, partially funded for Greg Crawford’s DNAse Hypersensitivity studies. Both instruments are available for use by the Duke community.
Projects in the works for the Genome Analyzers include work from the Crawford lab, as well as David MacAlpine’s study of Okazaki fragments, and Greg Wray’s Costa Rica project and comparative gene expression between humans and chimps.
To plan your Illumina Sequencing project, go to Steps to Running Your Illumina Sequencing Project.
The IGSP Sequencing and Genetic Analysis Facility offers a number of other services including the following: a Biomek FX Liquid Handling Robot, a Bio-Rad Experion Automated Electrophoresis Station, a HydroShear Apparatus (Genomic Solutions), a Qiagen TissueLyser, and a Beckman Z-2 Particle Counter.