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The Genomics Shared Resource (GSR) is a multifunctional, state-of-the-art core laboratory that provides instrumentation and expertise to facilitate cancer research at OSUCCC – James.
As a support service, the GSR trains researchers how to use specific software and analyze data for any methods used in the GSR, in order to educate and enable them to analyze and manage data more effectively and to reduce the need for costly outside analysis. Additionally, the GSR assists researchers in methodology optimization to increase data quality and lower the costs where applicable.
The GSR offers the following support:
NOTE: For information on providing an appropriate quality of DNA and RNA, please refer to the following document: "DNA-RNA Isolation Considerations When Using TruSeq Library Prep Kits."
Digital Multiplexed Gene Expression Analysis Using the NanoString nCounter System
- Copy Number Variation Analysis: The nCounter Copy Number Variation CodeSets provide everything needed to interrogate up to 800 regions of the human genome in a single multiplexed reaction with as little as 300ng purified genomic DNA of starting material.
- nCounter Human Karyotype Panel: The Human Karyotype Panel consists of 338 probes spanning all 24 chromosomes at a rate of approximately 8 probes per chromosome arm.
- nCounter miRGE assay: Designed to simultaneously measure expression levels of both miRNAs and mRNA in a single reaction from a single sample (choose between 5-30 miRNAs of their choice to profile and between 100-200 mRNAs).
DNA Sequencing (Sanger-Based and Non-Sanger-Based)
Despite the new next-generation sequencing technologies, the demand for traditional sequencing is growing. No single method, however, can meet the needs of all studies. Therefore, along with the Illumina HiSeq 2500, a next-generation sequencing platform, the GSR offers an additional sequencing platform.
Sanger-Based DNA Sequencing. Traditional Sanger-based sequencing is a strong and cost-effective validation tool for genetic and epigenetic mechanisms involved in cancer development that are extensively studied at OSUCCC.
Most applications include targeted re-sequencing of genetic alterations such as mutations, deletions, duplications, larger structural rearrangements of cancer genes, animal modeling in cancer and biomarker identification. A variety of samples are processed daily, such as BACs, pure plasmids, mini-prep plasmids, M13 clones, cosmids, lambda clones, PCR products, gel-isolated fragments or bacterial genomes.
The GSR operates two 48-capillary Applied Biosystems 3730 DNA Analyzers: one for long-read and one for short-read applications and all the necessary accessory equipment for Sanger-based DNA sequencing. A modified Sanger dideoxy chemistry, the ABI Prism BigDye Terminator Cycle Sequencing Kit version 3.1 is the method of choice. Both machines use data collection software v3.0 and sequencing analysis software v5.2. Turn-around time is the same day or next morning, depending on priority, and the GSR Web site provides a sequencing request form with all the necessary information that routinely results in the highest-data quality.
One thousand nucleotides of sequence data can be routinely obtained from one long-read sequencing run in the form of a raw sequence data file and a chromatogram data file. Researchers initiate the process by submitting a specific amount of template combined with the primer along with a DNA Sequence Request Sheet denoting the type of reaction to be performed and primer and sample names as outlined on the GSR website.
Non-Sanger-Based DNA Sequencing
The lllumina HiSeq 2500 sequencer has undergone major changes since its launch in February 2010. This includes larger flow cells and new image analysis software to bring sequencing output to 1.6 billion (200 million reads per lane, eight lanes per flow cell) passed filter reads. In our first HiSeq 2000 run, some of our samples achieved 240 million reads per lane or 1.9 billion reads per flow cell. The new sequencing chemistry (version three) significantly improves coverage uniformity by reducing density-dependent GC bias and by increasing in cluster density, thereby resulting in the lowest number of gaps and minimal risk of missing variants in sequencing data. The Illumina primary alignment software algorithm CAVASA_v1.8.2 significantly improved SNP, indel and structural variant calling and increased aligned reads by 5 to 7 percent. The amount of mismatch-free reads per run has also increased threefold since 2010. Illumina achieved major breakthroughs in data-processing software (RTA 1.12), resulting in a significant reduction in the disk space required to store the sequencing output. For example, the amount of space needed to store HiSeq 2500 data from a human genome sequenced to a depth of 30-fold has dropped by an order of magnitude. Despite the huge amount of data output, network bandwidth needed for data transfer from the HiSeq 2500 instrument to our computation server is minimal (5.56 out of typical 100 mbs sustained capacity over a 1 gbs network).
The increase in sequencing output and the reduction in sequencing time enabled by the Illumina HiSeq 2500 Sequencer allows researchers to sequence more samples, increasing the statistical power and quickening the time to data while staying within their research budgets. For example, with the current data output, 125 indexed samples can be sequenced per run (two concurrent HiSeq flow cells at about 25 million reads per sample) for high-throughput gene expression profiling study. If the intent is to perform high resolution transcriptome analysis, more than 50 samples can be analyzed per run at about 100 million reads per sample. The run time for both types of runs is around two days.
Genotyping technology has undergone dramatic efficiency and speed changes because of ever-growing analysis needs for enormous numbers of polymorphisms in multiple samples. Many robust methods emerged with fast and efficient mechanisms for screening large populations for genetically linked traits and for cancer-related genes discovery. The GSR provides several different genotyping platforms.
Genotyping services include:
- Real-Time PCR-Based Assays
- Capillary Electrophoresis-Based Assays
- Illumina HiSeq 2500
Real-Time PCR-Based Assays (ABI 7900)
The Applied Biosystems 7900HT Sequence Detection System uses fluorescent-based PCR chemistries to provide qualitative detection of nucleic acid sequences by end-point analysis. An allelic discrimination assay is a multiplexed end-point assay that detects variants of a single nucleic acid sequence. The presence of two primer/probe pairs in each reaction allows genotyping of the two possible variants at the single-nucleic polymorphism (SNP) site in a target template sequence. TaqMan® SNP Genotyping Assays from Applied Biosystems provide a highly flexible technology for detection of polymorphisms within any genome. These TaqMan assays are routinely used for genotyping applications, including screening, association, candidate region, candidate gene or fine-mapping studies.
Capillary Electrophoresis-Based Assays (ABI 3730)
The GSR routinely uses the ABI SNaPshot® Multiplex System for SNP analysis on one of the two 48-capillary Applied Biosystems 3730 DNA Analyzers. The SNaPshot Multiplex System is a primer extension-based method that allows multiplexing of up to 10 SNPs. For SNP screening and confirmation, the PCR products are diluted and combined with the GeneScan™-120 LIZ® Size Standard to indicate the size of the labeled fragments. After the run, the GeneMapper® Software analyzes the data and generates allele calls. Other common screening methods for cancers include Micro Satellite Instability (MSI) for colorectal cancer and Loss of Heterozygosity (LOH) for breast cancer using microsatellite markers.
Illumina HiSeq 2500
Genotyping in a selected segment of the genome can be achieved using DNA-seq, exome or focused amplicon panels. This application is used for creating sequencing libraries from purified genomic DNA for subsequent analysis on the Illumina cBot and HiSeq 2500.
DNA Methylation Analysis
The GSR provides several different platforms for DNA methylation analysis: the Capillary Electrophoresis-Based Assays (ABI 3730) and the Illumina HiSeq 2500 System. Depending on the number of genes and samples being examined, the specific platform can be chosen and assigned.
Capillary Electrophoresis-Based Assays (ABI 3730)
Bisulphite conversion of unmethylated Cytosine is an easy and widely accepted method for detecting methylated CpGs. Sequencing bisulphite-treated DNA is the gold standard technique for methylation detection.
Bisulphite treatment (the bisulfate conversion of unmethylated Cytosines) of templates is usually performed by the investigator; the GSR performs methylation analysis using bisulphite sequencing. Additionally, the GSR can use SNaPshot® to quantitatively detect the base differences in treated and untreated samples to access the methylation status of test samples.
Illumina HiSeq 2500
DNA methylation status analysis can be achieved by the DNAMethylation-seq protocol. The application is for whole genome DNA methylation analysis, and the protocol is based on the bisulfite conversion method to study all sites of methylation in the entire genomes. Methylation profiling can also be done by sequencing the meDIP pulldown material or Diagenode AutoMethylCap kit.
Quantitative Real-Time PCR
Quantitative PCR (qPCR) is used for DNA quantitation such as viral load. Most applications use qPCR for the quantitation of RNA levels—more specifically, reverse-transcription quantitative PCR (RT-qPCR)—to measure the levels of mRNAs, miRNAs and other RNA species.
The GSR operates four Applied Biosystems 7900HT Fast Real-Time PCR Systems. Key applications include SNP detection using the fluorogenic 5’ nuclease assay (see genotyping) and gene expression quantification (absolute and relative). TaqMan™ and SYBR Green chemistries are supported.
Quantitative Real-Time PCR includes:
- TaqMan Gene Expression Assays
- TaqMan Low Density Array (TLDA)
- TaqMan MicroRNA Assays
- Digital Multiplexed Gene Expression Analysis Using the NanoString nCounter System
- Validation of Microarray Results
- SYBR Green Chemistry
- Data Analysis
TaqMan Gene Expression Assays
This method capitalizes on a quantitative relationship between the amount of starting target sample and the amount of PCR product at any given PCR cycle number.The 5’ nuclease chemistry exploits the exonuclease activity of AmpliTaq Gold DNA polymerase by using a cleavable fluorescent probe in combination with forward and reverse PCR primers. TaqMan Assays have the highest specificity, highest sensitivity and the largest dynamic range of any gene expression technology.
TaqMan Low Density Array (TLDA)
For more throughput, TaqMan Gene Expression Assays are available pre-loaded onto the TaqMan Low Density Array, a 384-well microfluidic card that enables one to eight samples to be run in parallel across 12 to 384 targets, without the need for liquid handling robotics.
Additionally, the GSR uses arrays customized with genes, pathways or prognostic panels with as few as 12 genes or as many as 380. Combined with a pre-amplification step, very small amounts of samples, as well as archived tissue samples such as laser-capture microdissection (LCM), formalin-fixed paraffin-embedded tissues (FFPE) and needle biopsies, can be processed for gene expression analysis.
TaqMan MicroRNA Assays and Arrays
TaqMan MicroRNA Assays incorporate a target-specific, stem-loop, reverse-transcription primer. The new primer design addresses a fundamental problem in miRNA quantitation caused by the short length of mature miRNAs (~22nt), which prohibits conventional design of a random-primed RT step followed by a specific real-time assay. The stem-loop structure provides specificity for only the mature miRNA target and forms a RT primer/mature miRNA-chimera that extends the 5’ end of the miRNA.
The resulting longer RT amplicon presents a template amenable to standard real-time PCR using TaqMan Assays. These assays are not only specific for mature miRNAs, but also can distinguish among highly homologous targets.
TaqMan assays are known for a wide linear dynamic range, up to nine logs, and a miRNA profile can be generated in as little as three hours compared to several days for microarrays.
TaqMan® MicroRNA Arrays provide all the advantages of TaqMan® MicroRNA Assays in a convenient, pre-configured microfluidic card minimizing experimental variability and effort required to run 384 TaqMan MicroRNA Assays in parallel.
TaqMan® MicroRNA Arrays v3.0 is a pre-configured 2 micro fluidic card set, A and B, with currently 754 unique assays specific to human microRNAs updated to capture new content available in Sanger miRBase v14. In addition, each array contains four control assays, three selected candidate endogenous control assays and one negative control assay. An optional preamplification step can be included for small amounts of sample.
Digital Multiplexed Gene Expression Analysis Using the NanoString nCounter System
NanoString’s novel digital technology uses color-coded, fluorescently labeled molecular barcodes and single molecule imaging to detect and count hundreds of unique transcripts in a single reaction. Bypassing the need for amplification and reverse transcription eliminates enzymatic bias and simplifies the nCounter workflow to consist of hybridization, post-hybridization processing and digital data acquisition. The complexity of the barcodes, containing one of four colors in each of six positions, allows a large diversity of targets present in the same sample to be individually distinguished during data collection.
The GSR has operated the nCounter System from NanoString Technologies since January 2010. In addition, by the end of March 2010, the OSUCCC GSR completed beta testing for a new product for microRNA analysis that uses multiplex digital barcode technology to profile the human miRNA transcriptome in a single tube.
The current applications include:
Gene Expression Analysis: NanoString’s gene expression CodeSets are pairs of approximately 50-bases called capture and reporter probes that hybridize to the RNA sample and are used to detect and count mRNA transcripts. The reporter probe carries the signal and the capture probe is used to immobilize the complex for data collection. After hybridization, samples are transferred to the nCounter Prep Station where excess probes are removed and probe/target complexes are aligned and immobilized in the nCounter Cartridge. Cartridges are then placed in the nCounter Digital Analyzer for data collection. This technology can detect and count hundreds of gene transcripts simultaneously with a sensitivity of less than one copy per cell.
The system can directly assay tissue and blood lysates as well as FFPE extracts with protocols starting from 100ng or less of total RNA. mRNA expression analysis can be performed simultaneously on up to 800 genes for validation and routine testing of whole-genome screens, for the quantification of entire pathways, biological processes, or prognostic signatures in a single tube.
miRNA Expression Analysis: The NanoString miRNA assay uses an additional sample processing step to enable the detection of small RNAs. Preparation of small RNA samples involves the ligation of a specific DNA tag onto the 3’ end of each mature miRNA; these tags are designed to normalize the Tm’s of the miRNAs as well as to provide a unique identification for each miRNA species in the sample. The tagging is accomplished in a multiplexed ligation reaction using reverse-complementary bridge oligonucleotides to direct the ligation of each miRNA to its designated tag. Following the ligation reaction, excess tags and bridges are removed, and the resulting material is hybridized with a panel of miRNA:tag-specific nCounter capture and barcoded reporter probes. Following purification, each captured barcode is counted and tabulated in the nCounter assay. NanoString miRNA assay can be used for discovery experiments because the entire micronome can be covered. Both nCounter human and mouse miRNA expression assay kits are available. 100ng or less of total RNA per sample is used for one profiling reaction.
Copy Number Variation Analysis: The nCounter Copy Number Variation CodeSets provide everything needed to interrogate up to 800 regions of the human genome in a single multiplexed reaction with as little as 300ng purified genomic DNA of starting material. The nCounter Custom CNV Assay is based on the standard nCounter assay with two important additions: DNA fragmentation and denaturation. These two steps yield single-stranded targets for hybridization with nCounter probe pairs, the reporter and capture probes, following the nCounter workflow including hybridization, post-hybridization processing and digital data acquisition. Each CNV probe pair is identified by the “color code” generated by six ordered fluorescent spots present on the Reporter Probe. The Reporter Probes on the surface of the cartridge are then counted and tabulated.
nCounter Human Karyotype Panel: The Human Karyotype Panel consists of 338 probes spanning all 24 chromosomes at a rate of approximately 8 probes per chromosome arm. This coverage enables highly accurate confirmation of diploidy and identification of aneuploidies for each chromosome. The nCounter Human Karyotype Panel utilizes the same work flow as the nCounter Custom CNV Assay. Prior to hybridization, 600ng of genomic DNA is fragmented and denatured to yield single-stranded targets for hybridization with the Karyotype Panel Code Set. After hybridization, samples are transferred to the nCounter Prep Station where unhybridized probes are removed and probe/target complexes are aligned and immobilized in the nCounter Cartridge. Cartridges are then placed in the nCounter Digital Analyzer for data collection. Analysis of Karyotype Panel data is automated by the nCounter CNV Collector Tool provided free with the panel.
Validation of Microarray Results
Microarrays are based on the same principle as Northern blots, differential hybridization and other hybridization-based techniques. Hits from microarrays are validated for two reasons:
- To verify the observed changes to ensure that they are reproducible in a larger number of samples
- To verify the array results to ensure that they did not result from problems inherent to the array technology.
Additionally, the relatively limited dynamic range of fluorescent microarrays places limits on the technology with respect to sensitivity and specificity. Therefore, it is essential to use independent means to verify that the genes of interest are truly differentially expressed and to what extent.
SYBR Green Chemistry
SYBR Green is an intercalating dye that fluoresces upon binding to double-stranded DNA. Intercalating dyes are inexpensive and easy to use, and they can be used for any reaction because they are not sequence specific. Because they do not discriminate between specific sequences, however, they cannot be used for multiplexed analysis.
Analysis of real-time qPCR data can be either of absolute or relative levels. The majority of GSR’s analyses use relative quantitation, which is easier to measure and of primary interest to researchers examining disease states.
For absolute quantitation, an RNA standard curve of the gene of interest is required to calculate the number of copies.
For relative quantitation, comparative Ct is the most common method. The GSR performs basic analysis of individual qPCR or lower throughput experiments. The Biomedical Informatics Shared Resource analyzes high-throughput experiments including all TaqMan low density arrays using the Integromics RealTime StatMiner® software.
Additional Services and Accessories
- Quantitative measurement and quality control of RNA/DNA
- Non-fluorescence and fluorescence Imaging (Typhoon 9410 Imager)
- Densitometric scanning (Personal Densitometer SI)
- DNA/RNA Synthesis support
RNA/DNA Quantitative Measurement and Quality Control
The Thermo Scientific NanoDrop TM2000 spectrophotometer, which measures nucleic acid concentrations, provides highly accurate UV/Vis analyses of 1ul samples with high reproducibility, and it produces results for a full continuous spectrum from 220-750 nm that allows quality control.