MCW Mellowes Center Research Services

• Many sample types (blood, saliva, tissue, FFPE, fresh frozen, frozen embedded tissue, cells) can be utilized.
  
  
• Commonly used model species (human, mouse, rat, etc.) that are ready for analysis; consult for additional development.
  
  
• Raw fastq, BAM or other file types for direct intake to additional bioinformatic analysis.

Nucleic acids are the building blocks of all cellular life and can provide a wealth of information about the processes and functions of biological systems. Our team can extract RNA and/or DNA from a variety of sources, including blood, saliva, cells and tissue (fresh frozen or formalin fixed paraffin embedded), with either low or high-throughput protocols. Following the determination of concentration, we can provide quality assessments based on absorbance ratios or size fragmentation.

To visualize and assess the size and concentration of nucleic acids, the Mellowes Center utilizes a fluorescent labeling, capillary electrophoresis system. For isolated RNA and DNA, the instrumentation compares experimental samples to an internal ladder standard to estimate degradation status and concentration. Additionally, in preparation for next generation sequencing, shearing protocols and prepared libraries can be run to assess shearing or insert size and presence of any undesirable adapter or primer peaks.

Whole Genome Sequencing (WGS) is a method to comprehensively consider the DNA base pairs of a sample and evaluate variants that arise in the sequence. The role of genomic variants in disease phenotypes and outcomes can be further investigated with expression, molecular modeling, and epigenetic regulation studies. The flexible method allows for sequencing of any species of interest including, but not limited to, human, model organisms (mice, rat, etc.), plants, microbes, and more.

View Whole Genome Sequencing (WGS) test details (PDF)

Whole Exome Sequencing (WES) allows for the enrichment of ~2% of the human genome and focuses on the protein-coding regions that contain a large percentage of the known disease variants. Focusing on a smaller sequencing region allows for greater depth and maintains a concentrated approach to understanding the role of variants in disease phenotypes. Beyond categorization of variants with curated databases, research analysis can further investigate protein structure, dynamics, and function of the variant.

View Whole Exome Sequencing (WES) test details (PDF)

Transcriptomic Sequencing (RNA-Seq) provides scientists with an understanding of how a sample system responds to changes in conditions, treatments, gene variants, and a wide variety of other study designs. Measuring gene expression changes (differential expression, DE) can provide understanding of cellular and molecular mechanisms that underlie disease initiation, progression, response to environmental or therapeutic interventions and substantially more conditions. By identifying changes in gene transcript levels, transcript sequence and splicing patterns that ultimately may alter protein expression patterns, investigators can associate pathway activation to phenotypic differences across samples.

The Mellowes Center offers several preparations to accommodate investigator needs and sample quality:

• Bulk RNA-seq for high quality samples: Illumina library preparation for quality (RIN >7) and quantity (input >200ng) RNA
• Bulk RNA-seq for high quality, low input samples: Takara SMARTseq library preparation for polyA capture of mRNA with quantities <100ng
• Bulk RNA-seq for low quality, low input samples: Takara SMARTseq library preparation for rRNA degradation with quantities <100ng
• RNA panel capture: NanoString’s nCounter protocols for capture of 700+ panel of genes and quantification of transcript level

View Transcriptome Sequencing (RNA-Seq) test details (PDF)

Single Cell Sequencing (scRNA-Seq) provides scientists with an understanding of how individual cells respond to changes in conditions, treatments, gene variants, and a wide variety of other study designs. By sequencing as many as 10,000 individual cells per sample, rare cell types may be identified, and differential expression of cell clusters can be separated from a heterogeneous population. Mellowes Center will provide advice on nuclei isolation and quality control on cellular viability with independent investigators maintaining responsibility for troubleshooting the isolation protocol for viable single cell suspensions.

View Single Cell Sequencing (scRNA-Seq) test details (PDF)

Understanding the localization of transcripts to cells and structures within a tissue is the foundation of Spatial Transcriptomics. When the entire transcriptome can be mapped with morphological context to the tissue, investigators can uncover novel mechanisms in tissue development, disease pathology, and translational research. The Mellowes Center combines the technology of the 10x Genomics Visium platform with the CytAssist instrument for rigorous tissue alignment to provide consistent sample preparation (for formalin fixed paraffin embedded tissues) and analysis.

View Spatial Transcriptomic Sequencing test details (PDF)

MicroRNA (miRNA) plays an important role in controlling cellular gene expression and often create inhibitory feedback loops and act as negative regulators. To reveal the miRNAs present in cellular systems, the Mellowes Center utilizes NanoString’s hybridization method to match, barcode and count the miRNA transcripts present in samples. The panel identifies and can compare expression among conditions of up to 800 biologically relevant miRNA molecules (available as human or mouse panels).

View MicroRNA (miRNA) Expression Panel test details (PDF)

DNA methylation is an epigenetic mechanism whereby tissues and cells can regulate gene expression, specifically hypermethylation of promoters and intergenic regions typically leads to repressed gene expression. To focus on the methylation status of a small portion of the genome (1-5%), enzymes first target CpG regions and then utilize bisulfite conversion to identify methylated bases during next generation sequencing. Methylation changes have been linked to and play a critical role in cancer initiation, disease progression, aging, tissue development, and many other cellular processes.

View Reduced Representation Bisulfite Sequencing (RRBS) test details (PDF)

The Assay for Transposase Accessible Chromatin sequencing (ATAC-Seq) method is used to understand and map the epigenetic landscape (packaging in open, euchromatin or closed, heterochromatin spaces) of DNA in the cell. This assay evaluates changes in histone positioning and DNA accessibility, mapping the genomic locations and associated genes that change during disease progression, drug treatments, or other experimental conditions. Often used as a screening tool, this method can help researchers further focus on follow-up experiments that reveal expression changes in pathways or epigenetic marks influencing cellular phenotypes and disease systems.

View Assay for Transposase Accessible Chromatin Sequencing (ATAC-Seq) test details (PDF)

Chromatin Immunoprecipitation Sequencing (ChIP-Seq) is an epigenetic method to understand the intersection of proteins (transcription factors or histones with regulatory marks) with specific regions of the human genome. Understanding and mapping these interactions can reveal common binding sites and delineate structural changes that can regulate the ultimate ability of the cell to express various genes. By adding a layer of epigenetic regulation to the transcriptional alterations occurring in cells and tissues, investigators can bring additional mechanistic understanding to tissue development, disease pathology, and translational research. To reduce input needed and background signals, cut&run or cut&tag technologies may also be utilized during the immunoprecipitation protocol.

View Assay for Chromatin Immunoprecipitation Sequencing (ChIPSeq) Test Details (PDF)

The Mellowes Center has a wide array of NGS platforms and can work with individual investigators to align experimental needs with equipment available. During a consultation, investigators can discuss with our ‘Omics and BI teams the development of custom panels, novel captures, or unique library preparation techniques. In this way, we can partner and ensure the ability to sequence and analyze unique and focused preparations. Additionally, we are excited to partner with investigators to develop new protocols and generate new bioinformatic analyses that answer novel hypotheses, ultimately clarifying the mechanisms to a variety of disease states, environmental conditions, cellular responses, and much more.

View Custom Next Generation Sequencing (NGS) test details (PDF)

For those developmental or exploratory-based data analyses, such as analyzing new sequencing data types, integrating various data types or re-analyzing existing data to gain novel biological insights, development-based services are provided.

Costs are based on per-project rate, i.e. a certain percentage of a full-time effort (FTE) of a bioinformatics analyst. Rates range from 10%-40% FTE, with a minimum of six months.

The specific rate for a particular project should be discussed and determined through a consultation meeting.