Services

The ATG Shared Resource provides the highest-quality bioinformatics analysis of genomics data sets, employing state-of-the-art tools such as custom scripts written in "R". The ATG staff has analyzed data sets for dozens of laboratories and have produced high quality figures for numerous publications and grant applications. The ATG Facility provides a full range of data analysis services including:

• Pre-experiment consultation and assistance with experimental design issues related to genomics experiments
• Customized data analysis using filtering and statistical methods that make use of stringent quality controls
• Preparation of publication-quality figures for papers or grant applications
• Storage of data and assistance with up-loading data sets to databases for publication

We strongly recommend that all users contact the facility staff before beginning their genomics experiments. We can provide information about the number of replicates that are required, how the DNA or RNA samples should be prepared and what the total cost is likely to be. A few minutes of discussion may save weeks and many dollars later.

Gene expression assays measure which genes are expressed in different samples, or how the expression of genes change over time or in response to specific treatments. The ATG Shared Resource specializes in using RNA-seq for measuring gene expression.

Next-Gen Sequencing and RNA-Seq

RNA-sequencing (RNA-seq) is a next-generation sequencing approach to measuring gene expression across all the genes at once. Briefly, RNA is isolated, fragmented, converted to cDNA then ligated to adaptors and sequenced. The resulting sequencing "reads" are aligned to the genome. The number of reads aligned to each gene is proportional to the gene expression level. RNA-seq may also provide information about mutations (e.g. SNPs) and alternative RNA splicing, and chromosomal translocations can be detected by analyzing the output for the presence of fusion transcripts that cross the translocation breakpoint. RNA-seq can be successfully performed with high-quality RNA from fresh or frozen tissues or cell lines, and with RNA isolated from pathology samples, such as Formaldehyde-Fixed, Paraffin-Embedded (FFPE) material.

Genotyping assays measure allelic differences, especially differences in Single Nucleotide Polymorphisms (SNPs), which are the differences in the DNA between individuals. The ATG shared resource can measure allelic differences in up to 100 SNPs using TaqMan real-time PCR assays. Alternatively, Affymetrix whole genome SNP arrays can be used to follow nearly 2 million SNPs across the whole genome. The latter assays are intended for mapping phenotypes to particular regions of the genome, experiments known as whole genome association studies.

The Analytical and Translational Genomics Shared Resource is a Certified Exome Sequencing provider using the Ion Ampliseq Exome Sequencing system and the Ion Proton sequencing instrument. The Ion Ampliseq system is a targeted multiplex-PCR assay that amplifies >250,000 amplicons from all of the exomes covering protein-coding regions in the human genome. The highly multiplexed products are then converted into sequencing libraries for the Ion Proton. The assays are usually barcoded and run two at a time on a P1 chip, which typically produces about 80 million "reads" and >100x average coverage across the targeted regions (about 2% of the human genome), which is more than adequate to detect germline variants (SNPs). The Ion Ampliseq assay is very robust and is ideal for the analysis of family units (trios) or genetic markers linked to ethnicity.

The ATG staff provide several types of bioinformatics analysis for exome sequencing data, including some standardized pipelines provided by the Life Technologies Ion Reporter tool, as well as customized analysis pipelines utilizing R/Bioconductor tools.

The level of coverage offered by the Ion Ampliseq Exome assay may be sufficient for analyzing tumor samples, but because tumors are a mixture of tumor cells and normal cells, and may include subclones (e.g. tumor heterogeneity), this coverage may not be sufficient to detect all tumor variants, especially those present in only a subset of tumor cells (i.e. tumor subclones). For "deep" sequencing of rare subpopulations in a tumor or to characterize tumor heterogeneity, it is preferable to use a targeted assay such as the Comprehensive Cancer Panel.

The ATG facility provides targeted sequencing of >400 cancer-relevant genes using the Ion Ampliseq Comprehensive Cancer Panel, a multiplexed PCR-based assay that results in the sequencing of all of the protein-coding regions for nearly all oncogenes, tumor suppressors and other genes important in cancer biology. These assays are usually barcoded and run four at a time on a single P1 chip on the Ion Proton sequencer, which typically produces about 80 million "reads" and produces >750x average read depth across the targeted regions. That makes these assays ideal for detecting rare variants (e.g. in <5% of the tumor cells) or for studying tumor heterogeneity (subclones within the tumor). The CCP assay is very robust and works well with DNA from FFPE material or fresh or frozen tissue.

The ATG facility uses some standard bioinformatics tools to detect variants (SNVs) identified by the CCP assay, and has developed customized scripts in R/Bioconductor to analyze other features such as tumor heterogeneity, loss of heterozygosity (LOH), copy number variants, etc. However, these assays also require analysis of normal tissue for comparison. An excellent combination is to use the CCP targeted assay for the tumor tissue, combined with the Ion Ampliseq Exome assay for normal tissue to detect germline (inherited) variants and markers linked to ethnicity.

The Analytical and Translational Genomics Shared Resource is currently developing several types of epigenetics assays based on next-generation sequencing assays, including Chromatin Immuno Precipitation sequencing (ChIP-seq) for the analysis of histone modifications and transcription factor binding sites. Please contact Dr. Ness if you are interested in developing or using these assays in your laboratory.