Technologies

Applications: Whole Genome/Exome Sequencing, Gene Panel Sequencing, RNA-Seq (incl. Single Cell RNA-Seq), ChIP-Seq, Methyl-Seq, Microbiome Sequencing

With the power to sequence millions to hundreds of millions of DNA fragments simultaneously in a matter of hours or few days, NGS has been the major driving force in life science and medicine in the last decade. The sequences (or reads) generated from the DNA molecules can be used to assemble new genomes, while more often they are used for mutation or variation detection, differential gene expression analysis, detection of protein-DNA interaction, epigenomics, and environmental microbial diversity profiling.

Northwestern investigators continually pioneer new strategies to solve their research questions with NGS. To meet the sequencing needs of our users, NUSeq maintains a fleet of major NGS systems, summarized below. If your project requires a different NGS platform, please notify us as we are constantly working to build our capacity.

Applications: Full-Length Transcript Profiling (at both bulk and single cell levels), Splicing Isoform Detection, Fusion Transcript Detection, Whole Genome Assembly, Haplotype Resolution, Structural Variant Detection, DNA Repeat Expansion Determination, Native Methylation Calling, Metagenomics

Long read sequencing technologies overcome challenges and limitations of short-read sequencing, and enable investigators to resolve complex genomic regions, accurately assemble genomes, identify structural variants, and unravele gene alternative splicing isoforms. Below are two currently available long read sequencing platforms available at NUSeq, both of which have seen significant advancements in recent years and continue to improve on accuracy and throughput.

Applications: Single-Cell RNA-Seq, Single Cell ATAC-Seq, Single-Cell DNA-Seq, Single-Cell Methyl-Seq, Single-Cell Multi-Omics

Single cell sequencing offers unprecedented opportunities to study cell-to-cell variation, identify/visualize different cell types/identities in a population, and infer cellular developmental trajectories. NUSeq offers multiple single-cell sequencing platforms, including 10x Genomics and Parse Biosciences, to meet the increasing needs for single cell sequencing, As listed above, besides transcriptome, single cell sequencing increasingly encompasses genome, exome, and epigenome of individual cells. While most single cell sequencing projects analyze large numbers of cells, the Core also offers a low-throughput option for teams who need to sequence small numbers of cells at greater depth.

Applications: Spatially Resolved Transcriptomics, Tissue Neighborhood Analysis, Tumor Microenvironment Analysis, Cell-Cell Interaction Inference, Spatial Cell Atlasing

Spatial context is important for cells to carry out their functions. For diseased conditions, pathologists examine tissue sections to look for microenvironments where cells turn abnormal. Spatial genomics provides a “map” of gene activity in morphology-preserved tissues, often with subcellular resolution. This state-of-the-art technology enables us to investigate gene expression in the context of tissue structure to shed light on functional diversity and heterogeneity in regions of interest. Depending on technical approaches employed, spatial resolution of gene transcripts in tissue sections is achieved using a sequencing-based workflow or in situ hybridization.

Applications: Genome-Wide Genotyping, Epigenome-Wide DNA Methylation Profiling

Microarray was the first high-throughput genome technology developed based on hybridization of pre-designed nucleic acid probes to molecules present in user samples. Signal readouts from the hybridization are then used to analyze gene expression, gene sequence variation, DNA methylation, etc. Currently most NUSeq users employ this time-tested technology for genome-wide genotyping and methylation analyses. Illumina genotyping arrays can be used in genome-wide association studies (GWAS) to establish relationships between nucleotide/structural changes and development of diseases (or biological traits) in a variety of species. For epigenome-wide association studies (EWAS), the Illumina human methylationEPIC and mouse methylation arrays are used to identify differentially methylated cytosines at single nucleotide resolution across the genome.

Applications: Targeted gene expression profiling

The NanoString nCounter Sprint Profiler is a platform for multiplex analysis of up to 800 RNA or DNA targets. This platform is based on molecular hybridization using target-specific probe pairs, and the digital readout of each target is achieved by NanoString’s unique fluorescence barcoding and single molecule imaging. This system is designed for studies that interrogate gene expression in a particular pathway or biological process. In addition to the catalogued gene panels (or CodeSets) below, users have the option of designing custom panels based on specific needs. The system also enables detection of gene fusions using junction probes, analysis of miRNAs, and simultaneous measurement of DNA/RNA/proteins.

• Predesigned panels: Cardiovascular Disease, Cell and Gene Therapy, Immunology, Infectious Disease, Neuroscience, and Oncology
• Number of samples processed per cartridge: 12
• Input required: 50 ng total RNA if extracted from fresh cells or flash frozen tissue; 150 ng if from FFPE tissue. Low RNA input (needs amplification prior to hybridization): 1 ng if extracted from fresh cells or flash frozen samples; 10 ng RNA if from FFPE tissue.

Please visit the NanoString service page for more details.

Applications: Detection of DNA Target (e.g., ctDNA or pathogenic DNA), CNV, Target Gene Expression, CRISPR Genome Editing Screening, NGS Library QC, NGS Discovery Validation

Droplet Digital PCR (ddPCR)

Digital PCR is the latest generation of PCR technology for absolute quantification of target DNA/RNA molecule(s), with significantly improved sensitivity and accuracy. This technology is especially suitable for targeted detection of circulating tumor DNA in liquid biopsy, rare somatic DNA mutations, or pathogenic nucleic acids at levels below the current detection limit. The detection sensitivity can reach 1 mutation in 1 million human genomic DNA copies, or 1 bacterial/viral DNA copy in 500,000 human cells.

NUSeq maintains the Bio-Rad QX200 ddPCR System, which has the capability to generate 20,000 individual PCR reaction droplets from a 20 ul PCR reaction. After PCR, all droplets are examined by the droplet reader for presence or absence of amplification signal. The percentage of positive droplets are then used to calculate the absolute copy number of the target DNA/RNA molecule(s) in the original sample on the basis of Poisson statistics.

(above) The Bio-Rad QX200 ddPCR System and the steps of carrying out a ddPCR experiment.

Quantitative Real-Time PCR (qPCR)

NUSeq maintains a QuantStudio 7 Flex system (ThermoFisher) to meet users’ project needs for real-time PCR technology. This system provides interchangeable block formats (96-well fast, 384-well, and TaqMan array card block) and sufficient multiplexing capabilities. This system is equipped with streamlined software, responsive touch-screen, automation capabilities, and easy block change without the need for any tools.

Applications: Cataloged Cell Line Confirmation, Patient-Derived Cell Line Matching

Verifying the identity of a cell line used in a project is essential to maintain scientific rigor and experimental reproducibility. NUSeq achieves authentication of human cell lines through an STR (Short Tandem Repeats) profiling assay. By analyzing a relatively small number of STR loci, this assay provides enough power to discriminate different human cell lines.

NUSeq uses the GenePrint 24 System from Promega for our STR-based cell line authentication. This system is based on co-amplification and five-color detection of 24 STR loci as shown below.

Applications: DNA and RNA Extraction (prior to downstream analyses including sequencing, array processing, PCR, etc.)

Automated or Manual DNA/RNA Extraction

High-throughput DNA/RNA extraction is offered on automated platforms (see below) to accommodate projects that submit large numbers (e.g., thousands) of samples. For samples that need special attention, manual extraction is also provided. NUSeq has experience extracting DNA, RNA, or both (i.e., co-purification), from a variety of sources, including FFPE sections, flash frozen tissues, whole blood, buffy coat, serum/plasma, saliva, buccal swabs, and cultured cells.

Chemagic 360 from PerkinElmer (now Revvity)

• Based on chemagen’s M-PVA Magnetic Bead technology
• Ideal for larger volume samples like saliva or whole blood
• Input volume: up to 4 ml if liquid (blood, saliva, etc.)

Maxwell RSC 48 from Promega

• Uses Promega’s cartridge-based Paramagnetic Particle Moving technology
• Ideal for high-throughput projects with smaller sample sizes
• Input volume: up to 400 µl if liquid (blood, saliva, etc.)

Manual extraction is also available upon request. For more details on the Core’s DNA/RNA services, please visit the DNA/RNA Extraction Service page.

DNA/RNA Sample QC

Core-prepared or user-submitted samples are qualified and quantified using Bioanalyzer, TapeStation, Fragment Analyzer, and Qubit. Sequencing libraries are also quantified using qPCR. Please visit the DNA/RNA Quality Control page for more information.