Sequencing

Currently, the genome center runs 16 3730 DNA Analyzers, which are traditional Sanger sequencers. To complement the 3730 data, we utilize 2 different types of next generation sequencers: the Roche 454 and the Illumina GAIIx. The total capacity of the center is 7-8 million sequencing lanes per year.

DNA Isolation

Plasmids are inoculated into growth media and incubated in floor shakers for 14~16 hours. After the samples have grown overnight, two types of DNA extraction methods are used. For difficult samples where multiple sequencing attempts are needed and very high quality DNA is desired, the Qiagen R.E.A.L. Prep Protocol together with the Qiagen BioRobot is used to isolate DNA in a 96-well format. This is a version of the classic alkaline lysis method that extracts purified DNA with silica-based filters. All other projects in a 384-well format use the Sprintprep Protocol from Agencourt Biosciences, automated on a Beckman Biomek FX Robot. This isolation mechanism incorporates magnetic beads into the alkaline lysis protocal to yield purified DNA. The magnetic bead prep is a highly automated, high-throughput system that yields small amounts of good-quality DNA which we use for shotgun style sequencing projects.

DNA Sequencing

Purified DNA is brought to the sequencing station where a 96-head Hamilton transfers DNA into a 384-well PCR plate. Sequencing cocktail consisting of primers, DNA polymerase, magnesium chloride, ddNTPs and dNTPs are added using an Innovadyne Nanopipettor that dispenses the cocktail without touching the bottom of the well in precise small volumes. The PCR plates are sealed and placed onto an ABI PCR 9700 Thermocycler that amplifies the sample of interest using the added primer set in 60~100 cycles and incorporates 4 fluorescently labeled molecules, one each corresponding to the 4 DNA base pairs.

Collecting Data on 3730 DNA Analyzers

The cycle-sequenced, amplified products are cleaned up to remove unincorporated dyes using another magnetic bead based system called CleanSeq form Agencourt Biosciences. The samples are resuspended in water, placed into cassettes and loaded onto the 3730 DNA Analyzer from Applied Biosystems. Samples migrate through a polymer matrix filled capillary tube and then exit into a water bath where the 4 different colored fluorescent labels are excited by a laser and the resulting colored flashes are recorded using a CCD camera. The instrument records 96 lanes at a time and the resulting color intensities (called chromatograms) are translated into the As, Ts, Cs and Gs of DNA sequence.

Next Generation Sequencing

The Roche 454 and the Illumina GAIIx platforms use sequencing by synthesis technology to capture sequence data. In both models, the DNA is copied in successive cycles and images are taken during base addition.  In the case of the 454, the luciferase enzyme causes a light reaction upon base incorporation.  On the other hand, the GAIIx uses fluorescence, like the 3730, to detect base incorporation. These new types of sequencers can provide as much sequence data in one run as all 16 of the 3730 machines make running for an entire week! By utilizing all 3 types of sequencers, we are better able to complete genome projects in a more timely and efficient manner.