DNA Sequencing

DNA sequencing

Shared Resource Director: Phillip SanMiguel
E-mail: pmiguel@purdue.edu
Department: Horticulture and Landscape Architecture
Shared Resource Operator/DNA Sequencing Manager: Doug Murphy
Phone: (765) 496-7368
Building/Room: WSLR, Room S045
Shared Resource Operator/Bioinformatics Specialist: Rick Westerman
E-mail: westerm@purdue.edu
Phone: (765) 494-0505
Building/Room: WSLR, Room S049

General Information and Services

The objective of the DNA Sequencing Shared Resource is to provide Purdue University Center for Cancer Research researchers the following service: determination of the sequence of plasmid or PCR product DNA using a standard vector primer or a custom primer, and web-based delivery of the sequence, its chromatogram and BLAST searches. Those interested in utilizing this service should consult its web page: http://www.genomics.purdue.edu/LTL.  An Affymetrix GeneChip Analyzer was recently purchased and DNA Array Analysis will be an additional service provided by the Shared Resource.

Overview of the DNA Sequencing Shared Resource

The current DNA Sequencing Shared Resource is part of a newly created campus-wide DNA Analysis Facility that resulted from a reorganization and consolidation of various DNA analysis services on campus. The DNA Sequencing Shared Resource is housed in the sub-basement of the Whistler Building located across the street from the main Cancer Research Center building (Hansen).

Description and Management of the Facility

Sequencing is performed on an ABI 3700 sequencer. Plate centrifuges, a Biomek 2000 laboratory robot, 384-well thermal cyclers and various multi-channel pipetting devices facilitate this process. Back-end support for the sequencers is provided by a four-processor Sun Enterprise 450 with four megabytes of RAM and over 400 gigabytes of disk space. 100 megabit/second network connections link the DNA Sequencing Shared Resource with the Enterprise 450 and the rest of Purdue University. Rick Westerman, the bioinformatics specialist, maintains various genetics programs including the GCG and the EMBOSS package and has written new web-based software customized to the needs of this Shared Resource.

Samples are submitted, tracked and analyzed via custom web-based software developed by this Shared Resource. Sample DNA concentrations are validated using a DNAQUANT fluorimeter. Dye terminator reactions using custom primers supplied by the investigator or common vector primers stocked by the Shared Resource can be performed, purified and analyzed in a single day. The time between sample submission and sequence delivery is commonly two working days. Sequence quality is monitored and sequence reads not producing 400 high quality bases are repeated, using a different chemistry, if necessary.

Users of this Shared Resource are automatically notified via email of the availability of their sequence and associated reports. This email includes a hyperlink to a password-protected user webpage. Graphic displays of the sequence data (chromatograms) are converted to PDF and JPEG formats using VTRACE and are viewable using Adobe Acrobat or a browser. BLASTN and BLASTX searches are performed such that HTML links to GenBank are inserted for each hit. Raw text sequence, quality-clipped sequence and quality values for each base are included in the web report.

DNA services rates