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.

Illumina NovaSeq 6000

Flexible, multiple flowcell formats to meet needs on throughput and/or turnaround time; Generates 800 million – 20 billion reads (or read pairs), or 65 – 6,000 Gb data in 13 – 44 hours, up to 2x250 bp in read length; 58 human genomes/426 exomes/800 transcriptome per run

Illumina HiSeq 4000

5 billion single reads or 10 billion paired-end reads, up to 1500 Gbp sequence data in <1-3.5 days, up to 150 bp in read length; 12 human genomes/96 exomes/100 transcriptomes per run

Illumina NextSeq 500

400 million single reads or 800 million PE reads, up to 120 Gbp sequence data in 11-29 hours, up to 150 bp in read length; 1 human genomes/12 exomes/16 transcriptomes per run

Illumina MiSeq

25 million single reads or 50 million PE reads, up to 15 Gbp sequence data in 4-56 hours, up to 300 bp in read length; good for microbiome 16S rRNA sequencing, small genome, small RNA sequencing, targeted DNA or RNA sequencing and screening of CRISPR/Cas9-induced gene editing events

NUSeq offers library construction for the different NGS applications. For the entire list of library prep options, please visit the NGS service page. For pre-made libraries, the Core offers library QC, qPCR and ddPCR-based quantification, and size selection (for libraries that contain adapter dimers and/or >1kb fragments).

Applications: Genotyping, Genome-Wide DNA Methylation Analysis

Microarray was the first high-throughput genome technology developed in the mid-1990s. It was originally invented for gene expression profiling, but its application soon diversified into other areas, including genotyping and DNA methylation analysis. As attested by the large amounts of data deposited in major genomics databases such as Gene Expression Omnibus (GEO), microarray technology has significantly advanced high-throughput genome research for over two decades.

Microarray is 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 pattern, DNA sequence polymorphism, DNA methylation, etc. The NUSeq Core provides support to two of the most widely adopted microarray platforms: the Illumina BeadArray and the Affymetrix GeneChip systems. The Illumina BeadArray technology creates an array with specially prepared, probe-carrying microbeads, which self-assemble into microwells etched into a substrate. Affymetrix GeneChip uses DNA probes synthesized in situ using photolithography, a process similar to that used for manufacturing semiconductor chips.

The NanoString nCounter Sprint Profiler — a new addition to NUSeq (as of September 2020) — is a platform for multiplex analysis of up to 800 RNA or DNA targets. For common pathways or bio-functions, NanoString provides a number of ready-to-use, predesigned gene panels, including those for oncology, immunology, and neurobiology. In addition to the catalogued gene panels, NUSeq Core users have the option of designing custom panels based on specific needs. This system is ideal for studies that interrogate genes in a particular pathway or bio-function, or to validate expression pattern of a group of genes after global gene expression profiling with RNA-seq. The system also enables detection of gene fusions using junction probes, analysis of miRNAs, and simultaneous measurement of DNA/RNA/proteins.

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.

Automated DNA Extraction

High-throughput DNA extraction is offered on an automated platform (AutoPure) to accommodate projects that require large numbers (e.g., thousands) of DNA samples to be extracted, or those that require DNA extraction on an ongoing basis. Accepted sample types are blood, buffy coat, saliva, and buccal swab.

DNA/RNA Sample QC

Core-prepared or user-submitted samples are qualified and quantified using a variety of means, including Bioanalyzer, Fragment Analyzer, and Qubit.