Thomas Jefferson UniversitySidney Kimmel Medical College

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Equipment

Facilities

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The Jefferson Cancer Genomics Laboratory Shared Resource is housed on tenth floor of the Bluemle Life Science Building in the Kimmel Cancer Center and is directed by Dr. Fortina. The facility consists of 1800-sq. ft. of space and includes two separate small rooms (80 and 60 sq. ft.) for placement of equipment and a walk-in cold room all adjacent to the main laboratory.

Major Equipment

Applied Biosystems-SOLiD 5500xl System Automated Sequencer (x2)

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The Sequencing by Oligonucleotide Ligation and Detection (SOLiD) 5500xl system is a massively parallel DNA sequencer based on sequencing-by-ligation. Each lane on the 5500xl can be independently addressed and configured on one of 2 flow chips, allowing for multiple applications to be run simultaneously including - DNA sequencing (whole genome and targeted), RNA transcriptome including long- and short non-coding RNA, RNA ChIP to study RNA-protein interactions SNP discovery and screening and epigenetic analysis (CHiP-Seq). The method uses emulsion PCR to produce amplified products of single DNA molecules onto microbeads, which are then deposited into one of the 12 lanes on 2 flow chips. The chemistry is run similarly to the SOLiD 4 systems with primer annealing, hybridization, ligation, imaging, and cleavage. Strings of 50 data bits, encoded by color, are aligned to a reference genome to decode the DNA sequence. 

Applied Biosystems EZ Bead System

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Applied Bioystem EZ-Bead System - The SOLiD-based NGS requires the library fragments to be amplified on microbeads. This step is termed ePCR and is a polymerase-mediated reaction that produces many copies of each single library fragment to allow that sequencing reactions will produce sufficient signal for detection by the instrument's optical system. The EZ Bead System automates templated bead preparation with an approximate 80% reduction in hands-on-time. It is comprised of 3 modules: 1) Emulsifier in which the user prepares aqueous phase (library, beads and PCR Master mix) and loads premixed oil to generate an emulsion; 2) Amplifier in which using a bag filling station, the user pours the emulsion into a bag, then seals, mounts and thermo cycles to generate templated beads in bagged emulsion; and, 3) Enricher in which the user loads reagents and consumables and pour emulsion into bead break vessel to generate pre-enriched bead QC aliquot and enriched templated beads. The total run time is approximately 7.5 to 8 hours. 

Ion Torrent PGM System

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Th Ion semiconductor benchtop sequencers perform real-time measurements of hydrogen ions produced during DNA replication. Ion semiconductor chips employ a massively parallel array of semiconductor sensors, to directly translate genetic information (DNA) to digital information (DNA sequence). Ion Torrent™ Technology directly translates chemically encoded information (A, C, G, T) into digital information (0, 1) on a semiconductor chip. This groundbreaking technology enables rapid and scalable sequencing across a range of applications. Library construction and template preparation can be done in as little as 6 hours using the Ion OneTouch System. From 3Mb to 1GB of output can be generated on the PGM in 4.5hours run time. Single samples can be run on the Ion 314 chip and multiplexing can be achieved using the Ion 316 and Ion 318 chips. Multiple samples and applications can be run on the PGM system - including targeted resequencing and amplicon sequencing, microbial and mitochondrial sequencing, RNA sequencing, validation and assessment sequencing, as well as, copy number variation and epigenetic analysis. 

Chemagen Magnetic Separation Module I Chemagic

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The Chemagen "chemagic" automated extraction system and chemistry produce consistently high yields of genomic DNA of high purity, integrity and stability in a safe enclosed environment. The system is a cost-effective solution to the challenge of processing large numbers of large-volume samples without compromising the requirement for a high degree of consistency of yield, quality and stability of isolated genomic DNA. Unique features of the Chemagen system include: 1) magnetic bead technology; 2) 12/96 rod head capable of handling both large volumes up to 10 ml or small volumes down to 50 µl; and, 3) samples processing in 20 to 60 minutes. Protocols for genomic DNA extraction from whole blood samples and buccal swabs are available.

NanoDrop® ND-1000

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The UV-based spectrophotometer functions as a full-spectrum spectrophotometer over the range 220 nm to 750 nm. The instrument uses 1.0 to 2.0 µl of sample with lowest limit of detection of 2 ng/µl.

Agilent 2100 Bioanalyzer

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The Agilent Bioanalyzer is used for rapid analysis of RNA and DNA samples. The system permits accurate sizing and quantitation of samples within minutes of loading and consumes only small amounts of material. This unit is especially important in assessing RNA quality and amounts prior to cDNA synthesis and labeling for microarray applications. In addition to characterizing total RNA used as starting material, the Bioanalyzer is able to check the quality of the RNA probe generated, check the status during labeling and fragmentation, and measure the amount which should be used in hybridization.

Applied Biosystems 3730 DNA Sequencers

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The Applied Biosystems 3730 48-capillary electrophoresis unit performs hundreds of sequencing reactions per day. Runs in excess of 700 bases can be obtained in 1.0 hours and even longer runs can be obtained by varying conditions. Plasmids, large insert clones and PCR products of a variety of sizes can be analyzed using BigDye Terminator Kit Version 3.1 and cycle sequencing protocols. Reactions are purified of excess terminators using XTerminator Solution from ABI prior to injection onto the capillaries for separation and detection. Secondary peak calls for detection of SNPs can be facilitated by SeqScape 2.5 and Sequencher 4.8 software.

Applied Biosystems Model 7900HT System

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The ABI Prism 7900HT Sequence Detection System is a second-generation sequence detection system instrument designed for automated, high-throughput detection of fluorescent PCR-related chemistries. An automated plate handler allows for high-throughput processing of plates. The instrument is capable of real-time, end-point, and dissociation curve analysis of assays arrayed in one of three formats: 96-well, 384-well or in a micro-fluidic format and can be set up to perform overnight runs without the need of supervision. Thermal cycling also can be performed on the 7900HT for real-time collection of PCR data. Software is system specific (ABI Prism 7000 SDS Software 1.2.3f2 for the SDS-7000 machine and SDS 2.3 for SDS-7900HT machine).

Applied BioSystems 9700 Thermal Cyclers

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Seven 9700 thermal cyclers are available for use. The 9700 permits amplification reactions to be performed in small volumes.

Affymetrix GeneChip System

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The Affymetrix GeneChip System consists of the following components: a hybridization oven, a fluidics station, the GeneChip Scanner 3000 with autoloader and a computer which controls the fluidics station and reader, collects the data and performs the basic analysis of the intensity from the array. Images and cell intensity files are created in the GeneChip Operating Software (GCOS) v1.4. The GeneChip system can be used for gene expression, genotyping, resequencing and mapping studies. Third-party software packages for performing gene clustering and construction of self-organizing maps are available.

Affymetrix GeneChip System DX2

The Affymetrix GeneChip® System 3000Dx v.2 (GCS 3000Dx v.2) is Affymetrix' second-generation solution for seamless DNA and RNA analysis. The GCS 3000Dx v.2, which is 510(k) cleared and CE marked for in vitro diagnostic use, consists of the GeneChip® Scanner 3000Dx v.2, the GeneChip® Fluidics Station 450Dx v.2 and Workstation with Affymetrix Molecular Diagnostics Software (AMDS). AMDS allows for patient data security with user-friendly interfaces allowing reporting and analysis software to be used in conjunction with the software. Current chips available for use on the system are the Human Genome U133 Plus 2.0 arrays for gene expression analysis and profiling and the Human Mapping 250K Nsp and the Genome-wide Human SNP 6.0 chips genetic profiling analysis.