Nanopore Day, Paris - POSTPONED

Tue 31st March 2020

Université Paris Diderot, Paris, France

Hear about the latest tech updates from Oxford Nanopore Technologies, as well as talks from scientists using nanopore sequencing in their work. 

There is no delegate fee for this event. 

Your place at this event will be confirmed via email from events@nanoporetech.com. Spaces are limited and will be allocated on a first-come, first-served basis.

Confirmed Speakers include:

• Xavier Fonrose, CHU Grenoble Alpes
• Julien Masliah-Planchon, Institut Curie
• Magali Hennion, ENS Paris
• Marie-Christine Carpentier/Olivier Panaud, Laboratoire Génome et Développement des Plantes Perpignan
• Ahidjo Ayouba, IRD/University of Montpellier
• Rohia Allili, UMRS1269 - NutriOmics
• Virginie Chesnais, Life & Soft
• Laurent Jourdren, IBENS
• Anna Dolnik, Hopital universitaire de la Charité Berlin

Would you like to present your work at this Nanopore Day?

We are looking for early career researchers to give lightning talks at our upcoming Nanopore Day in Paris. Each successful applicant will have the chance to take to the stage and give an 8 minute presentation. Find out more by clicking on apply to present.

Among the classical examples of pharmacogenetics (TPMT, NUDT15, CYP2D6, CYP3A5, CYP3A4), the genetic variants described have been identified in exonic or intronic regions. Structural variations are also present, particularly for CYP2D6. These variations are sometimes combined with each other and result in distinct pharmacogenetic phenotypes. They require both the search for frequent or rare variants, the resolution of haplotypes and structural variations, and the search for intronic variations. Current genotyping methods are based on sequencing by amplification of the exome or multiple small target regions. This can lead to the absence of detection of intronic variants (CYP3A5 *3, CYP3A4 *22), structural variants or rare variants and in all cases to the absence of haplotype resolution. Long read sequencing of native DNA using the Oxford Nanopore MinION® sequencer seems to be an answer to this problem. Through various examples, we will see how long read sequencing technology can solve some pharmacogenetic problems and the progress that still needs to be made.

Medulloblastoma (MB) can be classified into four molecular subgroups (WNT group, SHH group, group 3, and group 4). The gold standard of assignment of molecular subgroup through DNA methylation profiling uses Illumina EPIC array. However, this tool has some limitation in terms of cost and timing, in order to get the results soon enough for clinical use. We present an alternative DNA methylation assay based on nanopore sequencing efficient for rapid, cheaper, and reliable subgrouping of clinical MB samples. Low-depth whole genome with long-read single-molecule nanopore sequencing was used to simultaneously assess copy number profile and MB subgrouping based on DNA methylation. The DNA methylation data generated by Nanopore sequencing were compared to a publicly available reference cohort comprising over 2,800 brain tumors including the four subgroups of MB (Capper et al. Nature; 2018) to generate a score that estimates a confidence with a tumor group assignment. Among the 24 MB analyzed with nanopore sequencing (six WNT, nine SHH, five group 3, and four group 4), all of them were classified in the appropriate subgroup established by expression-based Nanostring subgrouping. In addition to the subgrouping, we also examine the genomic profile. Furthermore, all previously identified clinically relevant genomic rearrangements (mostly MYC and MYCN amplifications) were also detected with our assay. In conclusion, we are confirming the full reliability of nanopore sequencing as a novel rapid and cheap assay for methylation-based MB subgrouping. We now plan to implement this technology to other embryonal tumors of the central nervous system.

Genome replication mapping methods profile cell populations, masking cell-to-cell heterogeneity. Here, we describe FORK-seq, a nanopore sequencing method to map replication of single DNA molecules at 200-nucleotide resolution. By quantifying BrdU incorporation along pulse-chased replication intermediates from S. cerevisiae, we oriented 58,651 replication tracks reproducting population-based replication directionality profiles and mapped 4,964 and 4,485 individual initiation and termination events, respectively. Although most events clustered at known origins and fork merging zones, 7\% and 22\% of initiation and termination events, respectively, occurred at dispersed locations previously missed by population methods. Thus, FORK-seq reveals the full extent of cell-to-cell heterogeneity in DNA replication.

Utilisation du MinIon pour le séquençage rapide sur le terrain du virus Ebola et pour la caractérisation fine des virus de l'immunodéficience humaine (HIV) et simienne (SIV Notre unité de recherche travaille sur l’émergence de pathogènes (VIH, SIV, Ebola, etc) à l’interface Homme/faune sauvage en Afrique et l’évolution de ces pathogènes une fois établis au sein de la population humaine. Pour répondre à certaines des questions posées par cette problématique, nous avons utilisé le MinIon de Oxford Nanopore avec le Native barcoding kit pour caractériser en quasi temps-réel les souches du virus Ebola responsables des deux dernières épidémies en République Démocratique du Congo (RDC). Nous avons également utilisé le MinIon pour caractérisé un isolat de SIV infectant un singe vert d’Afrique. Enfin, nous utilisons le MinIon pour étudier la complexité de l’infection par VIH-1, notamment par la caractérisation de virus recombinants.

Use of the MinIon for rapid field sequencing of Ebola virus and for the fine characterization of the human and simian immunodeficiency viruses Our research unit works on the emergence of pathogens (HIV, SIV, Ebola, etc.) at the human / wildlife interface in Africa and the evolution of these pathogens once established in the human population. To answer some of the questions posed by this problem, we used the Oxford Nanopore MinIon with Native barcoding kits to characterize in near real time the strains of Ebola virus responsible for the last two outbreaks in the Democratic Republic of Congo (DRC). We also used the MinIon to characterize an SIV isolate infecting an African green monkey. Finally, we use the MinIon to study the complexity of HIV-1 infection, especially by the characterization of recombinant viruses.

Bibliography 1: Ayouba A, Mbala-Kingebeni P, Keita AK, Vidal N, Lacroix A, Touré A, Muyembe-Tamfum JJ, Delaporte E, Peeters M, Ahuka-Mundeke S. New technologies for controlling emerging infectious diseases in Guinea and the Democratic Republic of the Congo: their role in response to the Ebola epidemic. Med Sante Trop. 2019 Nov 1; 29(4):362-365. doi: 10.1684/mst.2019.0942. 2: Augier C, Beyne E, Villabona-Arenas CJ, Mpoudi Ngole E, Peeters M, Ayouba A. Identification of a Novel Simian Immunodeficiency Virus-Infected African Green Monkey (Chlorocebus tantalus) Confirms that Tantalus Monkeys in Cameroon Are Infected with a Mosaic SIVagm Lineage. AIDS Res Hum Retroviruses. 2019 Oct 30. doi: 10.1089/AID.2019.0216. [ 3: Mbala-Kingebeni P, Pratt CB, Wiley MR, Diagne MM, Makiala-Mandanda S, Aziza A, Di Paola N, Chitty JA, Diop M, Ayouba A, Vidal N, Faye O, Faye O, Karhemere S, Aruna A, Nsio J, Mulangu F, Mukadi D, Mukadi P, Kombe J, Mulumba A, Duraffour S, Likofata J, Pukuta E, Caviness K, Bartlett ML, Gonzalez J, Minogue T, Sozhamannan S, Gross SM, Schroth GP, Kuhn JH, Donaldson EF, Delaporte E, Sanchez-Lockhart M, Peeters M, Muyembe-Tamfum JJ, Alpha Sall A, Palacios G, Ahuka-Mundeke S. 2018 Ebola virus disease outbreak in Equateur Province, Democratic Republic of the Congo: a retrospective genomic characterisation. Lancet Infect Dis. 2019 Jun; 19(6):641-647. doi: 10.1016/S1473-3099(19)30124-0. 4: Mbala-Kingebeni P, Villabona-Arenas CJ, Vidal N, Likofata J, Nsio-Mbeta J, Makiala-Mandanda S, Mukadi D, Mukadi P, Kumakamba C, Djokolo B, Ayouba A, Delaporte E, Peeters M, Muyembe Tamfum JJ, Ahuka-Mundeke S. Rapid Confirmation of the Zaire Ebola Virus in the Outbreak of the Equateur Province in the Democratic Republic of Congo: Implications for Public Health Interventions. Clin Infect Dis. 2019 Jan 7; 68(2):330-333. doi: 10.1093/cid/ciy527.

Intestinal microbiota and metabolic disease : Nanopore-based signature of dysbiosis in severe obesity Rohia Alili, Eugeni Belda, Phuong Le, Jean-Daniel Zucker, Edi Prifti, Karine Clément.

Humans live in symbiosis with a large microbial populations that colonize in particular their digestive tract and constitute the gut microbiota. Disruption of this symbiosis induces a qualitative and functional alteration of the gut microbiota thus causing a so called “dysbiosis” that could play a role in many human disease pathophysiology such as in obesity. Studies have shown that the gut microbiota can indeed link changes in eating habits and lifestyle with metabolic alterations and low-grade inflammations that characterize obesity. Moreover in some obese subjects but not all, bacterial diversity is reduced and this decrease has been associated with higher metabolic deteriorations when compared to subjects whose bacterial diversity is preserved. During obesity surgery (i.e. bariatric surgery), dysbiosis improved but only partially corrected. Dietary constituents could be interesting in the management of the microbial ecology associated to obesity and related pathologies. Our study aims to stratify obese patients according to their gut microbiota diversity. This could be of interest to precisely target these patients with adapted nutritional recommendations in general and in the context of bariatric surgery. We thus developed a sequencing technology based on the use of the MinION tool from Oxford Nanopore Technologies. We chose a method for collecting human stool to stabilize bacterial DNA at room temperature, and then optimized a method for extracting DNA. Finally, we improved the preparation of the sequencing library and carried out a first validation of this technology compared to two other technologies : "SOLiD" and "Illumina". We confirmed the potential of using this technique in the patient stratification based on their intestinal microbial diversity.

Estimation of transcript isoforms is a real challenge with short read sequencing. Today with Oxford Nanopore Technologies (ONT), our aim is to sequence full-length cDNA in order to directly access transcript isoforms. We will present our work to optimise lab protocols to produce nearly full-length cDNA transcript reads, our primary analysis quality control tool (ToulligQC) and our on-going work to identify and count transcript isoforms.

Classification of hematopoietic malignancies is based on World Health Organization (WHO) guidelines and requires assessment of genomic aberrations. While a number of cytogenetic and molecular markers can be accessed in the clinic within a narrow timeframe of 48 to 72h, genomic aberrations can be detected only after 5 to 7 days and require cultivation of tumor cells (metaphases/karyotype) or labor extense applications limited to known genomic markers such as fluorescence in situ hybridization. Using whole genome sequencing with Oxford Nanopore technology (ONT) we routinely create copy number variation profiles based on a 24h run using one flowcell. This results in 10-19 GB sequencing yield corresponding to 2.5-5 fold genome coverage, which is sufficient to resolve aberrations with resolution of 0.1-0.5 Mb and detect subclonal alterations. In parallel, the respective DNA sequencing allows capture DNA methylation patterns that are characteristic for tumor subtypes and can be further used for classification. To enable the discovery of additional structural variations, we have established a complementary transcriptome sequencing protocol to allow the analysis of fusion genes in a similar timeframe. Using direct cDNA sequencing with SQK-DCS109 kits, we can achieve up to 7 million reads on a single flowcell in 24h. This approach is sufficient for robust detection of gene fusions (usually resulting in 50-90 reads supporting each fusion) and to provide gene expression profiles which can also be used for additional disease classification. This combined genome-, epigenome- and transcriptome-wide ONT based sequencing workflow provides the opportunity to rapidly characterize hematologic malignancies at the molecular level, which is needed for improved genotype-based treatment strategies. Currently, we have tested our approach in a large variety of biobanked samples and validation of the method on newly diagnosed myeloid malignancies is ongoing.

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Nanopore Day

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