Agilent Microarray Scanner
The Agilent microarray scanner is a versatile instrument used for all microarray applications, including gene expression profiling, high-density comparative genomic hybridization (CGH), and DNA methylation.
Services: Standard core services currently include gene expression profiling using the Single Color, Quick Amp and Low Input Quick Amp labeling kits on the Agilent 4x44K microarrays. Other formats available and priced upon request. Total RNA samples should contain 10 to 200 ng in a volume of 1.5 µl. A quality assessment of RNA integrity should be made on the Agilent 2100 Bioanalyzer. Samples should have a RIN value of at least 7 before proceeding.
Nimblegen Microarray Scanner
The high-resolution (2 um) of the MS 200 scanner from Nimblegen combined with the 12-bay MAUI Hybridization station make this a powerful platform for Gene Expression, Comparative Genomic Hybridization (CGH) studies, DNA Methylation, and analysis of ChIP-chip arrays.
Tecan Freedom EVO 200
The Tecan Freedom EVO is an extremely powerful instrument for the automation of liquid handling operations to provide accurate, high-throughput processing with minimal hands-on requirement. The robot is equipped with 8 fixed tips, plate manipulator arm (ROMA), tube barcode reader, and Infinite 200 plate reader. It is ideal for all repetitive operations including, dilutions and plating, cherry picking (hit picking), normalizations, Pico-Green measurements, reaction setup, and sequencing reaction cleanup. Fully programmable, the Tecan Freedom EVO can be configured to meet virtually any liquid handling needs.
Qiagen BioRobot Universal
Automated DNA/RNA purification from a variety of sample sources is being accomplished with a Qiagen BioRobot Universal system. All Qiagen 96-well format kits (DNAEasy, RNAEasy, PAXGene blood DNA etc.) can be automated on this robot to achieve high-quality DNA/RNA extractions and consistent results. BioRobot Universal is also used to perform numerous liquid handling tasks including sample normalization , PCR reaction setup, PCR clean up.
The Autopure LS enables automated purification of genomic DNA from large-volume samples using proven Puregene® chemistry. Manual handling of potentially-infectious samples is minimized. High-quality, purified DNA is stable for long-term storage and suitable for archiving as well as any downstream application. For increased convenience and flexibility, samples can be processed in batches of 8 or 16. Easy-to-use Autopure software provides diagnostic and troubleshooting functions that enable users to view the state of the instrument. To simplify data collection, information from each run is automatically stored in an integrated, searchable database.
ABI 7900 RT PCR
Considered to be the “gold standard” in real time PCR, the 7900 from Applied Biosystems combines the flexibility in PCR chemistry with 96 or 384 well capacity.
ABI OpenArray System
The OpenArray system provides an efficient and cost-effective platform to perform high-throughput, real-time PCR assays. On a chip the size of a standard microscope slide, 3072 individual PCR reactions may be performed simultaneously using either Taq-Man, or SYBR chemistry. The high-density format eliminates the need for multiplexing so that performance may be optimized. The Accufill loading system takes only minutes to run and assures that samples are handled without fear of contamination. The system is ideal for gene expression and genotyping studies. Numerous pre-configured panels are available from Applied Biosystems (Click here) or researchers may create panels which are completely customized. For absolute quantitation of message transcripts, the OpenArray platform is perfect for running digital PCR experiments.
Illumina Bead Express
The BeadXpress reader by Illumina is ideally suited for medium-throughput, targeted genotyping projects, allowing 48, 96 or 384 multiplexed SNP assays using GoldenGate chemistry on Veracode beads with samples in the 96-well plate format. The Genetics Core can assist with assay design and submission to Illumina for assay production. GoldenGate genotyping generally requires two days of laboratory processing time. DNA should be submitted at a concentration of no less than 50 ng/ul.
The Agilent 2100 Bioanalyzer is a microfluidics-based platform for sizing, quantification and quality control of DNA and RNA on a single platform. Results are delivered within 30-40 minutes in automated, high quality digital data. The Lab-on-a-chip platform is easy to use, faster than gels, and highly reproducible. The Bioanalyzer is also invaluable for making quality assessments at critical steps in the preparation of samples for microarray analysis.
3730xl Capillary Sequencer
Capillary DNA sequencing at the Genetics Core is carried out on an Applied Biosystems, 96-capillary, 3730xl DNA analyzer, which is the most advanced capillary sequencing platform available today. High quality reads in excess of 800 nt can be routinely achieved with proper sample preparation.
Multiple levels of service are available to meet the needs of the National Jewish Health and University of Colorado Denver research community. Those preferring to run their own Big Dye reactions (versions 1.1 or 3.1) can submit them to the core in a dried, “instrument Ready” form. Alternatively, primer and template may be submitted in the proper volumes and concentrations. The core will perform cycle sequencing, reaction clean-up, and provide data in a timely manner. Typically BigDye Terminator v3.1 cycle sequencing chemistry is used unless other chemistries are requested. Data for instrument ready samples are available by 10:00 am the following business morning. Full service samples submitted by 10:00 am will also processed within 24 hours.
5500xl Series Genetic Analysis System
The 5500xl Series Genetic Analysis System is a highly accurate, massively parallel next generation sequencing platform that supports a wide range of applications. The instrument is capable of sequencing 100Gb of mappable data, with reads length of 75bp. Reads can be multiplexed using 96 barcodes for DNA applications and 48 barcodes for sequencing RNA applications. The flexibility of independently run lanes in the microfluidic flowchip and multiplexing capability allow you to run a single lane quickly or conduct multiple experiments in a single run. Fragment, Mate-Pair and Paired-end library construction is available to facilitate characterization of the entire genome, transcriptome and epigenome, with specific applications including targeted resequencing, whole genome resequencing, small RNA analysis, gene expression analysis, chromatin immunoprecipitation and methylation analysis.
Roche/454 GS-FLX Titanium
The Roche GS FLX Sequencer utilizes Pyrosequencing to produce millions of long reads with exceptional accuracy in a single run. The instrument is capable of sequencing 300-560 Mb, with reads lengths of 300-450 nt. Reads can be multiplexed using 12 or more individual ‘MID’ tags. Shotgun or Mate pair libraries spanning insert sizes of up to 20 kb can be constructed. A GS-FLX picotiter plate can be divided into 2,4,8, or 16 regions.
Ion Torrent Personal Genome Machine
The Ion Torrent Personal Genome Machine (PGM) pairs natural sequencing chemistry with semiconductor technology resulting in a revolutionary sequencing process without fluorescence, enzymatic cascades, or optics. When a polymerase incorporates a nucleotide into a strand of DNA, a hydrogen ion is released resulting in a pH change. Ion Torrent captures this process in a massively parallel way using DNA loaded Ion Spheres on a high density array of wells atop a proprietary Ion sensor.
The flexiVent is a computer-controlled mechanical ventilator that measures respiratory mechanics. It can be integrated with the Aeroneb ultrasonic nebulizer that directly mounts onto the unit and delivers broncho-reactive agents deep into the lungs to test AHR (airway hypertension). The flexiVent uses indirect measuring techniques that offers complete control over; timing of the challenge, the nebulization rate and the manner in which aerosolization is synchronized with ventilation. The combination of these controlled experimental conditions, the flexiVent’s advanced indirect measurement techniques, and customized computer programs results in highly reproducible measurements with outstanding accuracy.
Nucleic Acid Extraction
The Genetics Core is equipped with several instruments to assist in the extraction of DNA and RNA from blood and tissues. The Autopure LS from Qiagen fully automates the extraction of DNA from fresh and frozen blood, buffy coat, Oragene Kits, cell lysayes and more using the proven Pure Gene chemistry. Automation reduces the handling of potentially infectious samples. For high-throughput extractions the BioRobot Universal completely automates the processing of PAXgene-96, and other 96-well kits from Qiagen. The QiaCube provides walk-away automation of Qiagen’s mini-column format nucleic extraction kits. Personnel in the Genetics Core can assist you with any of these technologies.
Increasing the efficiency of sample handling can significantly improve a researcher’s ability to generate and analyze large volumes of data. Laboratory robotics in the Integrated Center for Genes, Environment, and Health are available to the research community at National Jewish Health and the University of Colorado Denver to assist with high-throughput sample processing. The Tecan Freedom EVO 200 is equipped with a LiHa arm and 8 fixed pipet tips, a plate manipulator arm (ROMA), Pos-ID barcode reader, and an on-deck Infinite plate reader. The instrument is fully programmable to carry out virtually all pipetting operations including reaction set-up, sample dilution and plating, hit picking, sample normalization and pcr product clean-up. The BioRobot Universal from Qiagen is also fully programmable for all pipetting operations. Equipped with a shaker and vacuum manifold, the robot is especially suited to automation of nucleic acid extraction kits. Contact the Genetics Core for assistance with these instruments.
Chain-terminator sequencing (or Sanger method after its developer Frederick Sanger) relies on the incorporation of dye-labeled, elongation terminating, dideoxynucleotides to produce a set of DNA fragments of known length and known terminal residue. Fragments are separated by capillary electrophoresis. The Genetics Core uses the 3730xl instrument from Applied Biosystems; a 96 capillary instrument which is considered to be the gold standard in capillary sequencing. Several levels of sequencing service are available at the Genetics Core to meet your needs. Contact Genetics Core personnel to learn more about our services and sample drop-off spots at National Jewish Health.
Nucleic Acid QC/QA
Determination of quantity and quality of both DNA and RNA are essential for subsequent analysis. The Genetics Core is equipped with the instrumentation necessary to evaluate your nucleic acid samples before proceeding to expensive, and time-consuming downstream applications. The Bioanalyzer 2100 from Agilent featuring Lab-on-a-Chip technology allows for quick determination of size and quantity of DNA fragments from 25 to 12,000 bp. Several types of RNA chips are available including those to evaluate small molecules and samples as dilute as 50 pg/ul. The RNA Integrity Number (RIN), one of the key quality assessments before running micro arrays and constructing NGS libraries, is determined using the bioanalyzer. The Core is also equipped with the NanoDrop 1000 and the 8-channel NanoDrop 8000. The NanoDrop instruments are cuvette-free spectrophotometers requiring only microliter volumes to perform UV absorbance spectra. The instruments are fast and simple to operate.
SNP genotyping is the process of measuring genetic variations in single nucleotide polymorphisms between members of a species by examining their DNA sequences. SNPs are single-base differences at specific loci within genome that are found in more than 1 percent of the population.SNPs are found in both coding and non-coding regions of the genome.The SNP variations in a DNA sequence can have a major impact on how humans respond to disease, environmental factors such as bacteria, viruses, toxins, and chemicals; and drugs and other therapies.There are a number of new, cutting-edge technologies used in genotyping at the National Jewish Health Genetics Core.
The genotyping method best suited to your project is primarily determined by the size of your cohort and the number of SNPs you wish to interrogate. TaqMan chemistry is commonly used for SNP genotyping. For small projects which can conveniently be processed in 96 or 384 well plates, the 7900 instrument from Applied Biosystems has been the preferred workhorse for many years. The OpenArray system uses TaqMan chemistry in a very high-throughput format. Projects which involve large cohorts or many SNP assays are ideally suited to the OpenArray platform. While the content of the OpenArray chips is completely customizable, many pre-configured assay panels are available from Life Technologies. When large populations must be assayed for numerous SNPs, the Bead Express instrument using Illumina’s VeraCode technology can be the most economical approach. Core personnel can help you decide which instrumentation is best suited to your experiment.
The Genetic Core has many options and instrumentation available to answer questions about gene expression. Gene microarray technology measures the activity of many, previously-identified gene targets by hybridization to high-density array slides. We have both Agilent and Nimblegen array scanners. Gene arrays from many species are available from both manufacturers. Real time PCR can be used to make quantitative evaluations of gene expression. Depending upon the size of your project, either the 7900 or the OpenArray system, both from Applied Biosystems, are well suited to qPCR experiments. OpenArray is also ideal for absolute quantitation of transcript levels using a digital PCR approach. Serial Analysis of Gene Expression (SAGE) is a method based on the use of Next Generation sequencing techniques. Genetics Core personnel can help you decide which methods are best suited for your specific project.
Next Generation Sequencing
This technology enables massively parallel sequencing of nucleic acid fragments. The Integrated Center for Genes, Environment and Health (CGEH) offers access to state-of-the-art resources and services to help investigators, students and the biotech community conduct and promote research in the field of genomics. Our fee-per-use services include several applications such as: Whole Genome Sequencing, Targeted Sequencing, ChIP-Seq, Whole Transcriptome, Methyl-Seq, and Exome sequencing.
We provide solutions from experimental design to library-prep, sequencing, and custom data-analysis. Our system portfolio of scalable throughput enables us to provide sequencing flexibility in a cost effective manner to researchers. We have a comprehensive portfolio of Next Generation Sequencing services using the SOLiD 5500xl, 454 GS FLX Titanium and Ion Torrent Semiconductor systems.
Whole Genome Sequencing
The Genetics Core has an established pipeline for studying any size genome from bacteria to large, complex plants and animals using shotgun reads alone or in combination with multi-span paired end reads (3 kb, 8 kb, 20 kb). The Genetics Core can perform straightforward de novo assembly to decode previously uncharacterized genomes using long high-quality reads from either the GS FLX Titanium or the Ion Torrent Sequencer. The Genetics Core offers access to technology to obtain comprehensive genome coverage and explores the full range of genetic variation by resequencing organisms with an available reference using short reads from the SOLiD5500xl.
Resequencing experiments are fundamental in basic and clinical research as scientists look for causative mutations within populations. The identification of common and rare variants in the human genome is essential to better understanding the etiology of complex human diseases. Targeted resequencing allows researchers to efficiently and cost-effectively focus on regions of interest, for instance the protein coding portion of the genome, the exome, or custom candidate regions from 50 kb to up to 60 Mb. Whether resequencing amplicons, enriching genomic regions or RNA through target capture methods, our core personnel can help you identify the most appropriate enrichment strategy to sequence your target in a medium to high throughput scale using either our Ion Personal Genome Machine system or our SOLiD 5500xl system.
Chromatin immunoprecipitation (ChIP) is a technique for identifying and characterizing protein–DNA interactions involved in gene regulation or chromatin organization. Historically, ChIP reactions were analyzed by PCR or microarrays. While microarrays provide a method for “global” ChIP analysis, direct sequencing of enriched fragments has proven more effective in determining the binding sites of proteins along the genome in an unbiased manner. In collaboration with Dr. Brian O’Connor, we have established a ChIP-Seq pipeline. We have been examining the epigenetic regulation of immunity in the context of disease via ChIP-seq. We have developed methodologies to examine the genomic positioning of transcription factors, histone modifications and nucleosomes. Through this process, strategies for building libraries with small populations of cells have been identified as well as strategies for integrative analysis combining ChIP-seq with RNA-seq. Cross-platform comparisons (ex. Solid 5500xl vs. Ion Torrent) of ChIP-seq capabilities with immune cells are also performed.
Whole Transcriptome Sequencing
Global transcriptome analysis is of growing importance in understanding how altered expression of genetic variants contributes to complex diseases such as cancer, diabetes, and heart disease. Analysis of genome-wide differential RNA expression provides researchers with greater insights into biological pathways and molecular mechanisms that regulate cell fate, development, and disease progression. A sequence-based approach is a very powerful tool for studying gene expression, having the advantage of querying known as well as previously unknown RNAs in a sample in a “hypothesis-neutral” discovery. This approach requires making cDNA copies of all RNA present in the sample, including sequencing them, mapping the sequences back to a reference genome, and deducing the structure using bioinformatic tools. At the CGEH we use our SOLiD5500xl pipeline to analyze novel transcripts, novel isoforms, alternative splice sites, rare transcripts and coding SNPs.
DNA methylation is a key epigenetic modification to cytosines, often in CpG dinucleotides. This modification has been frequently associated with gene silencing, but the precise role of DNA methylation in development and disease remains a mystery. Whether your are interested in studying genome-wide profiling of DNA methylation or tackling methylated regions using an enrichment approach, our CGEH core personnel can assist you in using the SOLID5500xl sequencing pipeline to unveil methylation patterns of CPG islands.
Our FlexiVent service is tailored to fit the customer’s needs. Custom templates can be designed with the FlexiVent’s software, prior to an experiment wherein specific perturbations can be added or deleted and the rate of breaths/min can be controlled. FlexiVent experimental design and data analysis support is available upon request. New drug and reagent pilots can be performed by the Core’s flexiVent Service as well as new anesthesia pilot studies. The FlexiVent software automatically calculates specific parameters such as resistance, elastance, and compliance from the data collected from different types of maneuvers. Administration of anesthesia prior to FlexiVent experiments and blood sampling is available. Tissue samples can be collected upon request. Please contact The Genetics Core for more information.