Technologies

  • 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.

    Applications
    • RNA-seq: mRNA, non coding RNA, small RNA profiling; mRNA-seq analysis from single-cell is also available
    • DNA-seq: re-sequencing (whole genome, exome or targeted -seq), de novo-sequencing
    • ChIP-seq
    • 4C-seq
    • MNase-seq
    • ...
    Protocols and input material recommendations
    Protocol 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 200 ng of total RNA is required. A special protocol can be applied for lower input quantity (down to few ng).
    • Single-cell RNA-seq is also available. Capture, lysis, reverse transcription and amplification of up to 800 single-cells can be done at a time on the C1 Single-Cell Autoprep station from Fluidigm.
    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 exome or large custom regions sequencing, enrichment is performed with the Agilent SureSelect technology.
    ChIP-seq
    • For classical library preparation, the Illumina TruSeq protocol is applied.
    • 5 ng (or even less) of IP material is required.
    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.
    Illumina HiSeq 4000
    Photo Illumina
    Illumina MiSeq
    Photo Illumina
    PerkinElmer
    Zephyr workstation
    Photo Caliper/PerkinElmer
  • 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
    • 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.

    • 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).

    • 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 recommendations
    Protocol 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.
    Affymetrix GeneChip instruments

    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
    • SNP genotyping

      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.

    • 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 recommendations
    Protocol 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.
    Illumina iScan
    Photo Lunar
  • 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).

    NanoString Technologies nCounter
    Photo NanoString Technologies
    nCounter detail
    nCounter barcodes
    Picture jurvetson - Flickr
    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 recommendations
    Input 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.
  • 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
    • 384-well plates

      Plates are processed on the 7900HT and QuantStudio 12K Flex systems from Thermo Fisher Scientific or the LightCycler 480 from Roche.

      These instruments are equipped with automated devices for plates loading. Two liquid handling robots (one Tecan Freedom EVO and one Nanodrop Express of Innovadyne) are included in the workflow for plates filling.

      These automatized stations combine flexibility and high-precision measurement.

    • 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
    Applied Biosystems
    PRISM SDS 7900 HT
    Photo Applied Biosystems
    Roche LightCycler 480
    Photo Roche
    Applied Biosystems
    QuantStudio 12K Flex
    Photo Applied Biosystems
    Input material recommendations
    Input 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
    Thermo Fisher Scientific
    QuantStudio 3D
    Photo Thermo Fisher Scientific
  • Other equipment

    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.

    Fluidigm C1 Single-Cell
    Auto Prep System
    Photo Fluidigm

    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 two 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.

    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

    • One Freedom EVO from Tecan.
    • One Nanodrop Express of Innovadyne.
    Tecan Freedom EVO 150
    Photo Tecan
    Innovadyne Nanodrop Express
    Photo Innovadyne