Functional Genomics - Services
DNA Microarray: The Functional Genomics Shared Resource supports three array formats: spotted arrays of probes printed in-house on glass slides; oligonucleotide arrays synthesized by Affymetrix; and arrays using Illumina bead technology. The facility offers comprehensive spotted microarray analysis including RNA extraction and quality testing, sample clean-up, linear amplification (when indicated and requested), labeling, hybridization, scanning, preliminary image analysis, quality analysis, data management, data storage and conservation and experiment analysis. A GeneMachines Omnigrid Arrayer and an Axon 4000B Scanner are used for spotting and scanning of spotted microarrays, respectively. In addition, the facility works with investigators to design, print, and quality test custom human or murine arrays. Spotted arrays include numerous positive and negative control features (for example, features with no nucleic acid), features representing several genes from A. thaliana, approximately 60 housekeeping genes, and features containing poly-A or human genomic DNA.
The Functional Genomics Shared Resource also uses Affymetrix GeneChip technology for monitoring global gene expression using commercially available arrays capable of analyzing expression changes across entire genomes. Over the past few years, most users have relied on this technology rather than spotted arrays, primarily due to user preference for the commercial Affymetrix product. Affymetrix expression arrays are available for gene sets from human, rat, mouse, Arabidopsis, Drosophila, yeast, E. coli and other organisms. In addition, RNA extraction and quality analysis services are available and use a 2100 Bioanalyzer from Agilent Technologies, as well as NanoDrop ND-1000, to assure the quality and quantity of RNA before labeling.
The facility has expanded their platform of services by including the Illumina BeadXpress Reader. This system supports the development of both single- and two-color assays, across genotyping, gene expression, methylation, and protein-based assays. The BeadXpress System comes with VeraScan software, which provides a user-friendly graphical interface to control the BeadXpress Reader, enabling users to load assays, perform real-time scans, and view scan data for up to 96 samples per run. The software features administrator control of setting user specific accounts and rights, configuration of default file locations, and security options, including alerts via email. It also offers customization of scanning protocols, enables acquisition and viewing of intensity data, and report generation.
In addition, as a way to validate microarray expression results, we now offer real-time PCR services using the Agilent Technologies Stratagene Mx3005P. To access these services, the requester submits purified total RNA samples. Protocols for preparing RNA are available from the Functional Genomics Core Laboratory web site (http://www2.umdnj.edu/arrayweb/index.htm). An RNA quality check using the Agilent 2100 Bioanalyzer is performed and RNA concentrations determined by the NanoDrop ND-1000 spectrophotometer. Experimental samples are submitted with a control sample for comparison.
In addition to custom and standard spotted cDNA arrays, the Functional Genomics Shared Resource offers cDNA microarray printing services for members who provide their own cDNA clones or oligonucleotides. The Shared Resource also offers high-throughput pipetting using a Beckman Coulter Biomek FX Liquid Handling Workstation, to enable users to manipulate their own clone sets, should they wish to do so.
A comprehensive quality control program is used by the facility. Every GeneChip or spotted array contains performance features, which are examined, compared with industry and baseline criteria and recorded. Data from arrays or GeneChips that do not meet rigorous pre-determined performance standards are repeated.
Finally, the Functional Genomics Shared Resource is dedicated to educating the University with regard to microarray techniques and their applications. By sponsoring a series of lectures, it is anticipated that the scope of cancer research will be increased.
Next Generation Sequencing: (Note: We anticipate these services to come on line in the course of 2011 and are, appropriately, not reflected in the tables of use in Section 10 of this narrative.) The services provided will vary depending on which instrument and facility is used for the particular analysis performed. The extraction of RNA, DNA from tissues, cell lines, or chromosome immunopreciptations for Next Generation sequencing does not require any special conditions. These services can be provided by the core or can be carried out by the investigators themselves. In either case, the Shared Resource will perform quality analyses before proceeding to the next steps. By contrast, sample preparation for the Next Generation sequencing platforms, including library preparation, emulsion PCR, etc., are highly technical processes which must be performed correctly to ensure adequate sequences reads. These services will be included in the cost of sequencing and will be performed by Shared Resource staff.
The Roche/454 FLX pyrosequencing system uses massively parallel sequencing-by-synthesis (SBS) technology. The main advantage of this technology is the ability to generate longer reads (~400 nt) than the Illumina and SOLiD and platforms, which is useful for DNA methylation analysis, resequencing, and genomic sequencing applications.
The Applied Biosystems SOLiD4 Analyzer is capable of generating up to 71GB of sequence per slide, or 141GB per run. The SOLiD system is based on sequencing-by-ligation chemistry. Emulsion PCR is used to amplify unique DNA fragments on beads using universal primers. Beads are then attached to a microscope slide and the DNA fragments are interrogated through iterative ligation of fluorescent oligonucleotide probes. Each oligonucleotide probe interrogates two adjacent nucleotides at a time and the pair of nucleotides is recorded as a specific color. This dual interrogation allows increased accuracy (0.2% error rate). Currently, the SOLiD generates 35bp or 50bp reads in single read, pair-end, and mate-pair configurations. The SOLiD slide system can be run in whole slides, quarters, or octets providing multiplexing for up to 768 samples per slide (or 1536 sample per run). The number runs (full slides) required on the SOLiD 4 instrument can be reduced using the barcoding approach for multiplexing eight samples per run. This level of multiplexing dramatically reduces costs while still providing adequate sequence coverage for many applications.
The Illumina GAIIx Genome Analyzer is based on solid phase amplification followed by SBS of randomly fragmented DNA. The technology involves attachment of a short DNA fragment to a solid surface called a flow cell. The DNA fragments are then PCR amplified to create clusters at a very high-density (>25 million DNA clusters per lane) on the surface of a transparent sequencing flow cell. Amplified fragments representing a cluster are then sequenced and imaged with each reaction step. As the sequencing reaction occurs, fluorescently labeled reversible terminators are added, imaged, and removed allowing for the following sequencing step. The Illumina platform generates a much greater number of reads (~200 million/run, 48GB/run) and at a lower cost, which is approximately a 20-200 fold increase in sequence information over 454 per run which generates ~1 million 400 bp reads per run. However, the key disadvantages are shorter read lengths (36-151mers) and a longer run time (3-14 days) than with the 454. Illumina’s SBS approach provides much higher accuracy (<1.0% error per base).
The appropriate choice of instrument and experimental approach will be determined by the investigators in consultation with staff from the Functional Genomics, Biometrics, and Bioinformatics Shared Resources, who will be engaged in all stages of experimental design, data acquisition, processing and analysis. In general, both the Illumina and SOLiD platforms are ideal for RNA-Seq, ChIP-Seq and methylC(BS)-Seq applications, due to their ability to generate millions of short reads per run. While the 454 platform generates longer reads (~400 nt), the key advantage of the Illumina platform is that it is capable of generating up to 200-fold more sequence data for 1/2 to 1/3 of the 454 cost. The Roche/454 FLX pyrosequencing system uses massively parallel sequencing-by-synthesis (SBS) technology. As noted above, the main advantage of this technology is the ability to generate longer reads (~400 nt) than the Illumina and SOLiD and platforms, which is useful for DNA methylation analysis, resequencing, and genomic sequencing applications. Thus selection of the appropriate approach will be determined on a case-by-case basis.
Raw data from the sequence analysis will be pre-processed to ensure quality control and transferred to the Bioinformatics Shared Resource for detailed analysis (including functional analysis) to elucidate the underlying biological processes giving rise to the observed data. Processed data will also be stored on one terabyte external hard drives provided to the investigators.