Next Gen Sequencing (NGS)
The Genomics Platform is equipped with one HiSeq 4000, one HiSeq 2500 and one MiSeq from Illumina.
This highly robust and accurate sequencing platform is based on massively parallel sequencing of million of reads using proprietary reversible terminator-based sequencing chemistry (see method description on Illumina website). Whereas the HiSeq is ideal for large scale sequencing applications, the Miseq is preferred for projects that need smaller data output.
The complete system includes:
- a Zephyr robot from PerkinElmer for nucleic acids purification,
- a cBot cluster generation system for the preparation of the DNA clusters,
- two HiSeq (4000, 2500) and one MiSeq for sequencing.
The system enables sequencing of 50 to 300 bases from one end (single read; SR) or both ends (paired-end; PE) of DNA inserts, including indexing options.
- RNA-seq: mRNA and long non coding RNA profiling; mRNA-seq analysis from single-cell is also available
- DNA-seq: re-sequencing (whole genome, exome or targeted -seq), de novo-sequencing
Before bringing samples to the platform, please submit them in the genomics Laboratory Information Management System (LIMS) to help sample processing, monitoring and data retrieval (uhts-gva.vital-it.ch).
Affymetrix GeneChip system
The platform is equipped with 2 complete GeneChip instrument systems, each including a hybridization oven, two Fluidics 450 washing/staining stations, and a 7G scanner.
The Affymetrix technology is based on targets hybridization with oligonucleotides synthetized directly on arrays by photolithography. Arrays contain hundreds of thousands of oligonucleotide probes packed at extremely high densities.
Gene expression analysis
For standard RNA-based applications (gene expression) Affymetrix proposes microarrays covering numerous species, including human, mouse, rat, as well as numerous widely used model organisms (details on Thermo Fischer Scientific website).
Arrays contain probes covering multiple exons (Clariom S or Gene arrays) or every exon (Clariom D or Exon arrays) with the aim of increasing sensitivity and enabling to capture alternative splicing events. Some older designs contain probes designed toward the 3’ of the transcripts.
New target preparation protocols enable RNA profiling on FFPE samples.
Unbiased transcriptome analysis and ChIP-on-chip
Tiling arrays are also available for human, mouse, rat and some additional model organisms. These arrays contain probes regularly spaced along chromosomal sequences, irrespective of genome annotation (repetitive sequences being excluded). Tiling arrays permit new analysis of the genome, such as unbiased transcriptome analysis and chromatin immunoprecipitation (ChIP-on-chip).
For high resolution cytogenetic analysis, CytoScan arrays are available, providing high resolution and specificity for CNV, LOH, cnLOH and relevant mutations in cancer genes (details on Thermo Fischer Scientific website).
The Platform is equipped with an Illumina iScan instrument coupled to the Autoloader2. Assays are robotized on Tecan Freedom EVO instruments to improve throughput and technical consistency. This complete benchtop solution for genetic analysis enables a wide range of sample throughput.
Illumina proposes a range of BeadArrays with various numbers of SNPs interrogated, depending on the requirements of the projects. After whole genome amplification, Infinium assays are used: amplified DNA is hybridized onto arrays containing oligonucleotides with 3′ end positioned directly adjacent or overlapping the SNP site. SNP detection is performed by single base extension using dual colored assays (details on Illumina website). Infinium products comprise BeadArrays covering up to 4.3 million of SNPs and numerous CNVs. Custom or semi-custom arrays can also be designed.
Illumina has developped a robust methylation profiling platform that provides quantitative methylation measurement at the single-CpG-site resolution. Thanks to the Infinium assays, up to 850’000 methylation sites can be analyzed at a time.
The nCounter from NanoString Technologies is designed to measure highly multiplexed targets (up to 800-plex in a single tube) using fluorescently labeled reporter probes. Through the use of a barcoding technology, direct counting of individual molecules is made possible with a very high dynamic range, sensitivity, reproducibility, specificity and without any enzymatic reaction.
RNA and DNA molecules can be measured.
The probes used are about 100 bases long. They are designed by nanoString bioinformaticians on the basis of RefSeq ID’s or provided sequences.
The complete system includes two instruments. The PrepStation (liquid handling robot) is used to purify ternary complexes obtained during the hybridization, to remove unhybridized probes and to immobilize the complexes into the cartridge for subsequent image acquisition. The nCounter (image acquisition instrument) is used to scan the cartridge and generate raw data (counts).
- mRNA, miRNA, long non coding RNA profiling
- CNV analysis
- ChIP analysis (ChIP-string)
- New: simultaneous gene and protein measurement (available for the commercial PanCancer Immune codeset only)
Similarly to Real-time PCR, probes for samples normalization must be included in the codeset.
Preassembled codesets interrogating specific pathways or complete miRNA panels (human and mouse), are also available.
The system is perfectly suited for degraded RNA samples (ex: FFPE samples).
Real-time PCR experiments are performed on two 7900HT SDS systems from Thermo Fisher Scientific and one Light Cycler 480 system from Roche. These instruments are equipped with automated devices for plates loading and are thus designed for high throughput, with high precision in measures and good flexibility. 2 liquid handling robots (1 Tecan Freedom EVO and 1 Nanodrop Express of Innovadyne) are included in the workflow giving rise to 3 fully automatized Q-PCR stations.
Q-PCR is based on the measurement of the accumulation of very short amplicons in real time. We currently propose 2 alternative detection methods:
- Sybr Green: a non-specific dye which binds double-stranded DNA and fluoresces when intercalated.
- TaqMan probes: short oligonucleotides complementary to amplicons which fluoresce during PCR (under degradation). TaqMan probes provide additional level of specificity compared to Sybr Green.
- Real-time PCR is used for expression analysis of a small set of genes.
- CNV analysis can be performed.
- End-point PCR for detecting single nucleotide polymorphisms (SNPs) is also available. This allelic discrimination requires the use of 2 TaqMan probes: one specific probe for each SNP.
The platform is equipped with the QuantStudio® 3D System from Thermo Fisher Scientific.
End point PCR using fluorescent-labeled probes (or Sybr Green) is done on chips containing 20K partitions. Chips are loaded on the ProFlex instrument. Imaging is done on the QuantStudio® 3D.
- Absolute quantification (for DNA or cDNA)
- Copy Number Variation analysis
- Rare allele detection and quantification
- Low target copy detection
C1 Single-Cell Auto Prep System
The Single Cell Auto Prep System from Fluidigm is a new high throughput approach to single-cell genomic applications (mostly RNA-based applications) allowing researchers to identify cell-to-cell differences within a population of cells.
The C1 Single Cell Auto Prep System allows cell capture, lysis, reverse transcription and amplification of 96 or 800 single-cell at a time. Combined with RNA-seq for whole transcriptome analysis (including allele specific expression) the C1 Auto Prep System addresses biological questions that have previously been impossible to answer. See further details on Fluidigm website.
2100 Bioanalyzer and 2200 TapeStation
We routinely use the Agilent 2100 Bioanalyzer and 2200 TapeStation for quality control of total, messager or small RNA and of DNA samples before any application. Using electrophoresis technologies (in capillaries for the 2100 instrument), these instruments rapidly inform about RNA integrity and DNA size fragments in a quantitative way, using much less material compared to traditional electrophoresis gel.
QuBit and SpectraMax Gemini XPS Fluorescence-Luminescence Reader
In order to quantify very low quantities of material (e.g. LCM samples, FACS-sorted cells or ChIP material), the Genomics Platform is equipped with 2 fluorimeters: the QuBit from Invitrogen (for low throughtput) and the SpectraMax Gemini XPS fluorescence-luminescence reader (for high throughtput) from Molecular Devices. These instruments are used to quantify with high precision and sensitivity nucleic acids in solutions without prior purification of the samples, using fluorescent specific dyes for RNA or DNA.
The Covaris technology is based on the Adaptive Focused Acoustic (AFA) process established as the DNA shearing method, the first step in the preparation of NGS DNA sequencing libraries. It ensures the integrity of nucleic acid samples providing high recovery of double-stranded DNA. This technology allows to precisely and accurately fragment DNA and RNA from 100 bp to 5 kb.