Our areas of expertise
The genomics platform offers services adapted to sample preparation as well as quality and integrity control of extracted biological material.
This tailored service will adapt to the sample type (frozen, fixed, liquid...) and to the type of molecules of interest (mainly DNA, RNA). The suggested approach will follow good lab practice and quality criterea established in partnership with Institut Curie’s Biological Resource Centre.
In addition to a recognised know-how for the preparation of nucleic acids based on limited quantity or even poor quality samples, the platform put in place the preparation of ultra high weight molecular DNA (>100kb).
If you’re interested in helping us improve our techniques, contact us!
Quality controls are put in place either by the platform or by the users themselves after training for a fully autonomous use (via the online reservation tool Openiris).
Equipment :
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Crysotat CM3050 Leica
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KingFisher, Thermo
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Nanodrop ND8000, Thermo
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Qubit 8 canaux, Thermo
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BioAnalyzer, Agilent Technologies
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Fragment Analyzer, Agilent Technologies
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Femto Pulse, Agilent Technologies
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Robot Assist Plus, Integra
Reservation : Open Iris
DNA analyses
Polymorphism variations and structural alterations of genomes are analysed with specific methods that are in constant evolution.
Sequence variations that lead to nucleotide polymorphisms (SNPs) can be analysed in a targeted way by qPCR Taqman genotypage or even with an STR analysis by Sanger sequencing. Large-scale genotypage analyses are currently carried out via the use of Thermofisher Axiom chips (400k-700k markers). More exhaustive analyses are possible, don’t hesitate to contact us to put new tools in place.
Sequence variations due to insertions, deletions, translocations, repetitions that can lead to gains, losses of genomic regions, aneuploidies can be characterised with the help of high throughput molecular combing such as that offered by optic mapping developed by Bionano even by the use of SNP chips. The optic mapping approach can be subject to a specific development to mark regions of interest in a supplementary fluorescence canal. A collaboration would probably be established. If the characterisation of genomic structural variants requires further tools in particular if this is due to the complexity of anomalies of the studied genome, we offer targeted sequencing methods via Oxford Nanopore Technologies tools (gRNA/cas9 targeted sequencing, adaptive sequencing).
According to users’ requirements, the platform can prepare libraries for either sequencing or targeted sequencing.
Finally, primary data analysis can be led by the genomic team according to the expectations of teams, the level of knowledge required and the tools that we have available.
Targeted genomic analysis
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qPCR TaqMan (Open Array, ThermoFisher) genotyping
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Sequence repeats genotyping (STR, Promega)
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Identification of structural variants by optical mapping (Bionano)
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Characterization of structural variants by targeted Nanopore sequencing (adaptive sequencing, guide RNAs/Cas9).
Pangenomic analysis
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Genomic analysis of fresh or frozen samples using Cytoscan chips, or of fixed or degraded samples using Oncoscan chips (ThermoFisher)
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High-throughput genotyping using Axiom technology (ThermoFisher)
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Identification of structural genome variants by optical mapping (Bionano) of fresh or frozen samples
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Custom analysis by optical mapping (Optical replication mapping, Optical repair mapping, methylation, etc.).
Equipment dedicated to genomic analysis :
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Affymetrix ThermoFisher GCS3000 platform
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Affymetrix ThermoFisher Gene Titan 4C platform
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Applied Biosystems Real-Time PCR Systems: QuantStudio 12K flex and QuantStudio 5
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Accufill robot
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Saphyr system, Bionano
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MinIon, Oxford Nanopore Technologies
RNA analyses
Gene expression is complex and can be addressed at multiple levels. Coding RNAs for proteins can be quantified with the help of Thermofisher chips for a pan-genomic analysis by interrogated tens even hundreds of probes spread over the exonic and intronic regions.
qPCR or NanoString multiplexing approaches can be tailored depending on the type, the number of samples, the quantity of RNA available and the number of markers to quantify.
The platform also offers to analyse the regulation of gene expression by quantifying microRNA and non coding RNA by Thermofisher chips or by NanoString multiplexing.
The platform can test and put in place protocols dedicated to library preparation for transcriptome analysis by RNAseq.
Targeted transcriptomic analysis
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by Nanostring multiplexing approaches
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by quantitave PCR approaches
Non targeted transcriptomic analysis
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by Thermofisher chips interrogating transcripts coding or not for proteins (Clariom D)
Gene expression regulation analysis
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MicroRNA analysis by Thermofisher chips or by Nanostring multiplexing
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Analysis of DNA methylation using targeted approaches (HRM) or whole genome (preparation of WGBS, OxBS libraries)
For the evaluation of new methods of genomic analysis, don't hesitate to contact us !
Developments
The platform is committed to implementing and developing new techniques to meet the needs of teams.
Optic mapping (Bionano) was set up in the lab in January 2020 and put into service in June 2020. It allows the detection of large-size genomic structural variants such as insertion, deletion, translocations, repetitions, gained or lost regions, aneuploidies...
Developments for the steps 1, 2 and 3 in the above image are currently underway.
Indeed, several DNA extraction methods are being evaluated to address issues of quality and quantity of very high molecular weight DNA obtained with the Bionano method, from brain tissues and pigmented tissues. Double marking tests are also being tested.
Circosplot from a Bionano analysis of a uveal melanoma sample:
Long fragment targeted sequencing with Nanopore technology
ONT (Oxford Nanopore Technologies) was set up in the lab in January 2022. This technology allows us to sequence a whole genome, to target and enich a part of the genome using mainly an RNA guide and a Cas9 enzyme. The genomic team can take care of the design and test of this targeting sequencing approach. A selection of regions of interest by bioinfo enrichment in real time or adaptive sampling can also allow us to target the sequencing and to increase the coverage of regions of interest.
Example of a Nanopore sequence after Cas9 enrichment and adaptative sampling of the BAP1 gene of interest in uveal melanoma :
Molecular signatures and international collaborative projects
Depuis 2008, la plateforme a acquis un savoir-faire dans l’évolution d’outils d’analyse génomiques au bénéfice de la médecine moléculaire (médecine de précision, médecine de prédiction de risques), en participant à la mise en place de circuits biologiques s’inscrivant dans des essais cliniques. Ce savoir-faire a permis d’être un acteur majeur au sein de consortium nationaux et internationaux.
Since 2008, the platform has acquired expertise in the evolution of genomic analysis tools for the benefit of molecular medecine (precision medecine, risk prediction medecine) by participating in the establishment of biological circuits that are part of clinical trials. This expertise has allowed us to be a major actor in national and international consortia.
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Remagus04 trials (Institut Curie, Saint Louis Hospital, Gustave Roussy) - 2008 – analysis of the expression of 30 genes involved in chemotherapy response – Affymetrix Technology - Hess et al. (2006) doi.org/10.1016/S1470-2045(13)70611-9
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SAFIR01 trial (UNICANCER) - 2011 – Orienting breast cancer patients towards the most adapted clinical trial according to genomic alterations – Affymetric Technology - Comparative genomic hybridization array and DNA sequencing to direct treatment of metastatic breast cancer : a multicentre, prospective trial. DOI: 10.1016/S1470-2045(13)70611-9
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SAFIR02 (UNICANCER) - 2014 - validation of targetable genomic signature, breast and lung cancers - Affymetrix Technology. Nature. 2022 Oct;610(7931):343-348. DOI: 10.1038/s41586-022-05068-3. PLoS Med. 2016 Dec 27;13(12):e1002201. DOI:10.1371/journal.pmed.1002201.
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SHIVA (Institut Curie) - 2013 – analysis of genomic alterations – Affymetrix Technology - Molecularly targeted therapy based on tumour molecular profiling versus conventional therapy for advanced cancer (SHIVA): a multicentre, open-label, proof-of-concept, randomised, controlled phase 2 trial DOI: 10.1016/s1470-2045(15)00188-6
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PLoS Med. 2016 Dec 27;13(12):e1002201. DOI: 10.1371/journal.pmed.1002201.
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Oncotarget. 2017 Jan 3;8(1):1760-1773. DOI: 10.18632/oncotarget.12051
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Chin Clin Oncol. 2015 Sep;4(3):32. DOI: 10.3978/j.issn.2304-3865.2015.02.02.
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Classification COO (LYSARC) - 2015 – Classification of lymphoma subtypes from the expression of 20 genes - Nanostring technology - Reliable subtype classification of diffuse large B-cell lymphoma samples from GELA LNH2003 trials using the Lymph2Cx gene expression assay. DOI 10.3324/haematol.2017.166827
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Neopal trial - 2013 / Transneopal – 2020 - PAM50 signature – Classification and prediction of the risk of breast cancer relapse from the expression of 50 genes - Nanostring Technology - Prospective, multicenter French study evaluating the clinical impact of the Breast Cancer Intrinsic Subtype-Prosigna® Test in the management of early-stage breast cancers. DOI 10.1371/journal.pone.0185753
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MammoRisk (Predilife) : 2019 – analysis of genotypes predicting breast cancer - ThermoFisher Technology
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La Ligue/CIT program - validation of gene expression signatures - Nanostring technology
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LabEx Milieu Intérieur (Institut Pasteur) – Nanostring technology
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European MAARS project : 2014 – transcriptome analysis - Affymetrix technology