DNA Sequencing Facility
Core Description: The DNA Sequencing Facility provides reliable, long read, automated Sanger sequencing with fast turnaround; microsatellite-based genotyping and fragment analysis; plasmid and BAC DNA preparation and purification; and related molecular biological services including PCR, cloning, sub-cloning, site-directed mutagenesis, and preparation of targeting vectors for gene targeting in mice. It also provides services and support for analysis and interpretation of sequence data as well as the design of approaches to complex sequencing projects.
For the last four years the facility has been providing Roche 454 sequencing service that includes library preparation, emulsion PCR and pyrosequencing for both genomic DNA and amplicons. Data analysis is provided in each project depending on the investigator’s specific need.
Ion Torrent's Personal genome machine (PGM) is the latest addition at the facility. Known for scalability, simplicity and speed, this inexpensive technology is advancing fast to achieve new goals in terms of throughput and read length. The maximum read length and the throughput available at this point is 200 b and 1 Gb respectively. The applications are similar to those of long-read 454 sequencer and includes targeted resequencing of barcoded samples, sequencing of captured library, sequencing of bacterial and viral genomes, sequencing of metagenomic samples, RNA-seq specially small RNA sequencing and validation of sequence data obtained on other platforms. The sequencer comes with Torrent Suite, the Torrent server analysis pipeline that is the primary software used to process raw data acquired by PGM sequencer to produce sequence read files. The base calls are in both SFF and FASTQ file formats for easy downstream analysis with third party analysis tools. The Torrent suite performs filtering, trimming, mapping with the generation of a Variant Caller report. This long read sequencer is going to bring down the cost of new generation sequencing significantly.
The range of services mentioned above along with the expertise of the facility personnel enables this core to provide full support for investigators at Penn, who can easily obtain fast, reliable data on genes of interest, whether they are doing targeted or whole genome tumor genome sequencing, deep resequencing, screening clones for sequences of interest, establishing the identity of new clones, or searching for mutations in specific genes.
Tapan Ganguly, Ph.D.