Technologies
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Next Gen Sequencing (NGS)
Illumina
The Genomics Platform is equipped with the NovaSeq 6000 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).
The system enables sequencing of 100 to 250 bases from one end (single read; SR) or both ends (paired-end; PE) of DNA inserts.
Oxford Nanopore Technologies
The Platform is also equipped with the MinION Mk1C of Oxford Nanopore Technologies. This portable device for real-time DNA and RNA sequencing generates ultra-long reads (longest > 4 Mb) while keeping bases modification information.
This nanopore based sequencing system allows direct sequencing (without amplification or cDNA preparation) if needed.
Applications
- SARS-CoV-2 whole genome sequencing using the amplicon-based Paragon Genomics panel.
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RNA-seq: mRNA, non coding RNA, small RNA profiling. Isoforms, splice variants and fusion transcripts analysis.
mRNA-seq from single-cell is also available, allowing the identification of cell-to-cell differences within a population of cells. High-throughput analysis is available with the Chromium Controller system of 10x Genomics. - DNA-seq: genome assembly, re-sequencing (whole genome, exome or targeted -seq), structural variants identification.
- ChIP-seq
- ATAC-seq, MNase-seq
- Direct detection of DNA and RNA modifications
- 4C & HiC-seq
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Main protocols and input material recommendationsProtocol used and input material
RNA-seq
- For classical library preparation, the Illumina TruSeq protocol is applied.
- Poly A selection or ribo-depletion strategy can be applied to get rid of ribosomic RNA.
- For classical protocol, a minimum of 100 ng of total RNA is required. A special protocol can be applied for lower input quantity (down to few ng).
- A protocol of RNA exome capture is also available; ideal for low-quality samples such as FFPE samples.
- Single-cell RNA-seq is available.
DNA-seq
- For whole genome sequencing, Illumina TruSeq or Nextera sample preparation protocols are applied. 50 ng of DNA input is required, or 1 ng for smaller genomes.
- For targeted sequencing, enrichment is performed with IDT, Agilent or Twist captures.
ChIP-seq
- For classical library preparation, the Illumina TruSeq protocol is applied.
- Just a few ng are needed.
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. -
Microarrays
The Genomics Platform is equipped with the Affymetrix and the Illumina systems.
Affymetrix GeneChip system
The Platform is equipped with two 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.
Applications
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Gene expression analysis
For standard RNA-based applications (gene expression) including miRNA, Affymetrix proposes microarrays covering numerous species, including human, mouse, rat, as well as numerous widely used model organisms (details on Thermo Fisher 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.
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Constitutional cytogenetics
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 Fisher Scientific website).
Protocols and input material recommendationsProtocol used and input material
Gene expression
- For classical protocol, a minimum of 50 ng of total RNA is required.
- For lower input (down to 0.5 ng), a pico-kit is used.
Illumina BeadStation
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.
Applications
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Genotyping (GWAS)
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.
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Methylation analysis
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.
Protocols and input material recommendationsProtocol used and input material
Genotyping and methylation
- For whole-genome genotyping arrays and methylation arrays, 10 µL of DNA (un-treated or bisulfite treated, respectively) at 50 ng/µL are required.
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NanoString nCounter
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).
Applications
- mRNA, miRNA, long non coding RNA profiling
- CNV analysis
- ChIP analysis (ChIP-string)
- 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).
Input material recommendationsInput material
RNA
- For human, 100 ng of RNA is required. For bacteria, 10 ng is sufficient.
- Protocols of pre-amplification (requiring enzymatic reactions) can be used for low input material.
DNA
- 50 ng for bacteria to 600 ng of DNA for human are required for CNV analysis.
- 300 ng of ChIP material is required for ChIP-string.
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Real-time PCR
Real-Time (also called Quantitative-) PCR is based on the measurement of the accumulation of very short amplicons in real time. We currently propose two 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. Such probes can also be used for allelic discrimination by Q-PCR (one probe per allele is needed).
Real-Time PCR format
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384-well plates
Plates are processed on the QuantStudio 12K Flex system from Thermo Fisher Scientific.
This instrument is equipped with an automated device for plates loading. Three liquid handling robots (Tecan Freedom EVO) are included in the workflow for plates filling.
This automatized station combines flexibility and high-precision measurement.
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OpenArrays
OpenArrays are microscope slide–sized plates with 3’072 through-holes of 33 nL of volume, designed for Q-PCR. These assay-preloaded arrays are processed on the automated QuantStudio 12K Flex OpenArray AccuFill™ system (details on Thermo Fisher Scientific website).
The OpenArray technology helps streamline genotyping and expression studies that use large numbers of samples, assays, or both. Custom and commercial panels are available.
OpenArrays are probably the best-fit system for pharmacogenomics testing.
Applications
- Expression analysis, including miRNA detection
- Genotyping (= SNP detection = allelic discrimination)
- CNV analysis
Input material recommendationsInput material
- Hundreds of ng are sufficient (depending on the number of tested genes).
- For lower input quantity, protocol of pre-amplification can be applied.
Digital PCR
The aim of the Digital PCR is to quantify DNA molecules. It uses multiple PCR reaction partitions. Samples are diluted down to a limiting quantity such that most individual PCR reactions contain either zero or one target molecule. After PCR completion, the system counts the number of positive PCR wells that is the direct reflect of the number of molecules present.
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.
Applications
- Absolute quantification (for DNA or cDNA)
- Copy Number Variation analysis
- Rare allele detection and quantification
- Low target copy detection
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Other equipment
Single-cell systems
Single-cell analysis addresses biological questions that have previously been impossible to answer. The platform is equipped with two systems.
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Chromium Controller
The high throughput Chromium Controller system from 10x Genomics allows profiling of tens of thousands of single cells at a time thanks to the Gel Bead-in-Emulsion (GEM) technology (gel bead partitions or vesicles in which barcoded cDNA is prepared). The preparation of NGS libraries is then carried out in bulk reaction (see further details on 10x Genomics website).
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C1 Single-Cell Auto Prep System
The C1 Single-Cell Auto Prep System from Standard BioTools (former Fluidigm) is based on microfluidic technology. It is a mid-throughput approach allowing the capture of 800 single cells in individual chambers where cDNA amplification is carried out (see further details on Standard BioTools website). cDNA of 40 cells are then pooled before NGS library preparation.
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 Flex fluorimeter
In order to quantify very low quantities of material (e.g. FACS-sorted cells, ChIP material, ...), the Genomics Platform is equipped with the QuBit Flex fluorimeter from Invitrogen. This instrument is able 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.
Covaris
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.
Pipetting robots
- Three Freedom EVO from Tecan.
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