# Setting boundaries: Cohesin and mutually exclusive expression of virulence genes in the human malaria parasite (PlasmoVarOrg) - Mid term version DMP

Jessica M Bryant

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Jessica M Bryant. Setting boundaries: Cohesin and mutually exclusive expression of virulence genes in the human malaria parasite (PlasmoVarOrg) - Mid term version DMP. Institut Pasteur Paris. 2024. pasteur-04745591

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Submitted on 21 Oct 2024

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# "Setting boundaries: Cohesin and mutually exclusive expression of virulence genes in the human malaria parasite" project DMP

Plan de gestion de données créé à l'aide de DMP OPIDoR, basé sur le modèle "ANR - DMP template (english)" fourni par Agence nationale de la recherche (ANR).

## Plan Details

**Plan title** "Setting boundaries: Cohesin and mutually exclusive expression of virulence genes in the human malaria parasite" project DMP

**Version** Mid term version

**Fields of science and technology (from OECD classification)** Biological sciences (Natural sciences)

**Language** eng

**Creation date** 2022-04-04

**Last modification date** 2024-01-18

## Project Details

**Project title** Setting boundaries: Cohesin and mutually exclusive expression of virulence genes in the human malaria parasite

**Acronym** PlasmoVarOrg

**Abstract** Mutually exclusive expression of the var gene family is critical for infection and immune evasion by the deadliest human malaria parasite, Plasmodium falciparum. All but one var gene are silenced in heterochromatic clusters at the nuclear periphery, and activation is associated with spatial separation from this repressive cluster. Until now, the factors facilitating genome organization in P. falciparum were unknown; however, we recently found cohesin complex components enriched in var gene chromatin by mass spectrometry. Genome-wide analysis of a cohesin subunit revealed two peaks flanking a single var gene locus, suggesting a role in DNA looping or chromatin domain formation at the active var gene. The proposed project will use fluorescence microscopy, genome-wide approaches, and proteomics to provide an in-depth functional characterization of cohesin and explore how it functions in the organization and transcription of the var gene family, which impacts the virulence of the parasite.

**Funding**
* French National Research Agency : ANR-21-CE15-0010

**Start date** 2022-02-01

**End date** 2025-01-31

## Research outputs :

1. Images (Image)

2. Next Generation Sequencing (Dataset)
3. Mass spectrometry (Dataset)

Contributors

<table><thead><tr><td>Name</td><td>Affiliation</td><td>Roles</td></tr></thead><tbody><tr><td>Bryant Jessica</td><td></td><td><ul><li>DMP manager</li><li>Data contact (Images, NextGen Sequencing, Mass Spec)</li></ul></td></tr><tr><td>BRYANT Jessica</td><td>Institut Pasteur - Unité de Biologie des interactions hôte-parasite - 201021924S</td><td><ul><li>Project coordinator</li></ul></td></tr></tbody></table>
"Setting boundaries: Cohesin and mutually exclusive expression of virulence genes in the human malaria parasite" project DMP

# 1. Data description and collection or re-use of existing data

## Images

1a. How will new data be collected or produced and/or how will existing data be re-used?

All deliverables will come from original data acquired during the duration of the project. Images (immunofluorescent, western blot, DNA gel) will be generated with a microscope (Deltavision) and gel/blot imagers (BioRad ChemiDoc XRS), respectively.

1b. What data (for example the kind, formats, and volumes), will be collected or produced?

Images (immunofluorescent, western blot, DNA gel) will be captured in the manufacturer's format, but saved in .tif format. A maximum of 1,000 images at ~2MB each would equal 2GB of data.

## Next Generation Sequencing

1a. How will new data be collected or produced and/or how will existing data be re-used?

All deliverables will come from original data acquired during the duration of the project. Next generation sequencing data will be generated with an Illumina NextSeq500. Next generation sequencing data generated in this project may be compared to published data sets from our unit and other labs.

1b. What data (for example the kind, formats, and volumes), will be collected or produced?

Next generation sequencing data will be kept in fastq format. Approximately 250 libraries (ChIP-seq, RNA-seq, MicroC) will be sequenced, with approximately 2.5Gb of data per library (625Gb total).

## Mass spectrometry

1a. How will new data be collected or produced and/or how will existing data be re-used?

All deliverables will come from original data acquired during the duration of the project. Mass spectrometry data will be collected with a Thermo Fisher Q Exactive HF-X mass spectrometer in collaboration with the Laboratoire de Spéctrométrie de Masse Protéomique at the Institut Curie.
**1b. What data (for example the kind, formats, and volumes), will be collected or produced?**

The mass spectrometry data will be kept as .raw and .msf files, which are approximately 500Mb and 400Mb each, respectively. Approximately 32 samples (including replicates) will undergo mass spectrometry analysis, generating approximately 29Gb data total.

# 2. Documentation and data quality

## Images

**2a. What metadata and documentation (for example the methodology of data collection and way of organising data) will accompany the data?**

Each experiment performed in the lab has a specific identifier (the date the experiment was performed) and a title, and is explained in detail in an electronic lab notebook (eLabs). All raw data generated with an experiment is either attached (if an appropriate file type) to the experiment in the electronic lab notebook, or is kept in a folder in the lab's storage cloud (provided by the Institut Pasteur) that is labeled with the experiment identifier. Experimental details attached to data include parasite strain, stage of collection, how many parasites were collected, treatment of parasites, etc.

For imaging data, metadata (pixel size, bit depth, objective used, length of exposure, etc.) is stored in the file type generated by the equipment/software used to acquire the image. Although these files can generally be opened with Fiji, their corresponding images will also be saved as .tiff files to increase accessibility.

**2b. What data quality control measures will be used?**

All experiments will be performed with standardized protocols, which include appropriate numbers of replicates and controls. All raw data will be reviewed by the project leader.

Images (immunofluorescent, western blot, DNA gel) will be collected and manipulated minimally, in line with the ethics set forth by most respected peer-reviewed journals. Negative and positive controls will be performed along with test samples.

Microscopes are part of core facilities at the Institut Pasteur. They are maintained and/or calibrated professionally on a regular basis.

## Next Generation Sequencing

**2a. What metadata and documentation (for example the methodology of data collection and way of organising data) will accompany the data?**

Each experiment performed in the lab has a specific identifier (the date the experiment was performed) and a title, and is explained in detail in an electronic lab notebook (eLabs). All raw data generated with an experiment is either attached (if an appropriate file type) to the experiment in the electronic lab notebook, or is kept in a folder in the lab's storage cloud (provided by the Institut Pasteur) that is labeled with the experiment identifier. Experimental details attached to data include parasite strain, stage of collection, how many parasites were collected, treatment of parasites, etc.

Metadata attached to next generation sequencing data will meet the standards of the National Center for Biotechnology Information's Sequence Read Archive. Data files (.fastq) are linked to the following metadata: experiment (date and title), method (ChIP-seq, RNA-seq, ATAC-seq, etc.), sample (organism, strain, life cycle stage, control vs. knockdown, replicate number), and sequencing run (Illumina NextSeq500 2x150 base pair paired end). Published next generation sequencing data deposited in a repository will contain a digital object identifier linking it to the publication, which will provide an in-depth description of how the
data were generated experimentally and analysed.

**2b. What data quality control measures will be used?**

All experiments will be performed with standardized protocols, which include appropriate numbers of replicates and controls. All raw data will be reviewed by the project leader.

Sequencers are part of core facilities at the Institut Pasteur. They are maintained and/or calibrated professionally on a regular basis.

Next generation sequencing libraries will only be generated from experiments that have been validated by qPCR first and with
quality material. For example, RNA samples will be checked for degradation with a Bioanalyzer/TapeStation before being used to
make a library. Libraries will themselves be subjected to quality control using a Bioanalyzer/TapeStation and library quantification
before being sequenced. Quality of a sequencing run will be judged based on the sequencing reads, Illumina quality score
(FASTQC 30 or higher), and cluster density. Read mapping will be performed using bowtie2. PCR duplicates will be removed and
high quality (i.e. MAPQ score >= 20) reads will be retained using samtools. Appropriate sequencing depth will be achieved for
meaningful analysis. Genome-wide datasets will also be compared to published datasets. Technical and biological replicates will be
used in accordance with the ENCODE consortium data standards.

Mass spectrometry

**2a. What metadata and documentation (for example the methodology of data collection and way of organising data) will accompany the data?**

Each experiment performed in the lab has a specific identifier (the date the experiment was performed) and a title, and is
explained in detail in an electronic lab notebook (eLabs). All raw data generated with an experiment is either attached (if an
appropriate file type) to the experiment in the electronic lab notebook, or is kept in a folder in the lab's storage cloud (provided by
the Institut Pasteur) that is labeled with the experiment identifier. Experimental details attached to data include parasite strain,
stage of collection, how many parasites were collected, treatment of parasites, etc.

Mass spectrometry data files (.raw and .msf) are linked to the following metadata: experiment (date and title), method, sample (organism, strain, life cycle stage, control vs. knockdown, replicate number), and instrument of analysis. Published mass spectrometry data deposited on the PRIDE repository will contain a digital object identifier linking it to the publication, which will provide an in-depth description of how the data were generated experimentally and analysed.

**2b. What data quality control measures will be used?**

All experiments will be performed with standardized protocols, which include appropriate numbers of replicates and controls. For an immunoprecipitation-mass spectrometry experiment, five replicates will be performed for a control (strain that does not express an epitope-tagged protein) and test sample (strain that does express an epitope-tagged protein). Label-free quantitative comparison with the MyProMS software (https://github.com/bioinfo-pf-curie/myproms) will allow us to determine which proteins are significantly enriched in the test sample. All raw data will be reviewed by the project leader.

Mass spectrometers are part of a core facility at the Institut Curie. They are maintained and/or calibrated professionally on a
regular basis.

Raw mass spectral data files (.raw) are searched using Proteome Discoverer (Thermo Fisher) and Mascot (version 2.4.1) (Perkins et al, 1999). Mascot search parameters are: 10 ppm mass tolerance for precursor ions; 0.8Da for fragment ion mass tolerance; two missed cleavages of trypsin; fixed modification was carbamidomethylation of cysteine; variable modifications were methionine oxidation, CAMthiopropanoyl on lysine or protein N-terminal, and serine, threonine and tyrosine phosphorylation. Only peptides with a Mascot score greater than or equal to 25 and an isolation interference less than or equal to 30 are included in the data analysis.

**3. Storage and backup during the research process**
**3a. How will data and metadata be stored and backed up during the research?**

All lab notebooks are electronic (eLabs), which has been set up by the Institut Pasteur for scientific record keeping. In eLabs, experiments are organized by date and grouped by project. Data generated under a specific experiment will be stored in a folder with the same date and title as the experiment. All data will be stored on each group member's personal computer, as well as in eLabs (if the appropriate file type) or on the Institut Pasteur's secure internal server. In addition, data will be regularly backed up on external hard drives. Each staff member at the Institut Pasteur has 200 gigabytes of data storage available at no charge on the internal server, which is more than enough to cover the data storage needs of the proposed project. Once images, Next Generation sequencing, and mass spectrometry data have been published, they will also be stored in public and free databases (IDR, NCBI GEO, and PRIDE, respectively).

**3b. How will data security and protection of sensitive data be taken care during the research**

Our research does not concern human research subjects and contains no personal or sensitive information. During the research project, data will be stored in secure internal Pasteur servers, on eLabs, or on personal computers, all of which are only accessible with a password. External hard-drives containing backed up data will be kept in a locked drawer in the laboratory, which is only accessible with key card.

**4. Legal and ethical requirements, code of conduct**

**4a. If personal data are processed, how will compliance with legislation on personal data and on security be ensured?**

Our research does not concern human research subjects and contains no personal or sensitive information.

**4b. How will other legal issues, such as intellectual property rights and ownership, be managed? What legislation is applicable?**

All data generated under this project will be described in the eLabs electronic lab notebook (set up by the Institut Pasteur for scientific record keeping) and will be the property of the Institut Pasteur.

**4c. What ethical issues and codes of conduct are there, and how will they be taken into account?**

We will follow the Institut Pasteur, national, and international codes of conduct for scientific practice. There are no specific ethical guidelines or approvals needed to conduct the proposed project.

**5. Data sharing and long-term preservation**

**Images**

**5a. How and when will data be shared? Are there possible restrictions to data sharing or embargo reasons?**
This dataset will be made publicly available at the time of manuscript publication:

* analyzed data will be made publicly available in the publications. All manuscripts will be published open access, under a CC-BY-NC license.

* images associated with the publication will be made publicly available in Image Data Resource (IDR). All publications containing images will contain a project ID number for Image Data Resource (IDR).

During the project, the dataset will also be shared outside of the immediate team on a case-to-case basis (i.e. with colleagues or collaborators). My data sharing policy and links to all publications will be published on my laboratory website.

**5b. How will data for preservation be selected, and where data will be preserved long-term (for example a data repository or archive)?**

All conclusive and usable data will be stored during the maximum time allowed by the Institut Pasteur storage allowance capacity and the Image Data Resource (IDR). Data will also be stored in external hard disks in the laboratory.

**5c. What methods or software tools are needed to access and use data?**

As all images will be stored in .tif format, ImageJ, which is freely available, can be used to access and use the data.

**5d. How will the application of a unique and persistent identifier (such as a Digital Object Identifier (DOI)) to each data set be ensured?**

All images from a specific experiment and project will be compiled into a dataset folder with generated DOI from the data repository (IDR).

## Next Generation Sequencing

**5a. How and when will data be shared? Are there possible restrictions to data sharing or embargo reasons?**

This dataset will be made publicly available at the time of manuscript publication:

* analyzed data will be made publicly available in the publications. All manuscripts will be published open access, under a CC-BY-NC license.

* Next Generation Sequencing data (fastq or bedgraph files) associated with the publication will be made publicly available in NCBI GEO, a well-known repository for NGS data, and will be given a GEO reference number that will be linked in the publication. Sequencing data deposited on PlasmoDB is heavily used by the Plasmodium scientific community and is searchable by keyword or gene ID. GEO and PlasmoDB include extensive experimental metadata with files.

During the project, the dataset will also be shared outside of the immediate team on a case-to-case basis (i.e. with colleagues or collaborators). My data sharing policy and links to all publications will be published on my laboratory website.

**5b. How will data for preservation be selected, and where data will be preserved long-term (for example a data repository or archive)?**

All conclusive and usable data will be stored during the maximum time allowed by the Institut Pasteur storage allowance capacity, GEO, and PlasmoDB. Data will also be stored in external hard disks in the laboratory.
**5c. What methods or software tools are needed to access and use data?**

As data will be stored in `.fastq` format, these files can be opened with something as simple as text editor. Bedgraph files for viewing sequencing data can be opened and used in freely available software such as IGV or with websites such as UCSC genome browser.

**5d. How will the application of a unique and persistent identifier (such as a Digital Object Identifier (DOI)) to each data set be ensured?**

When published datasets are deposited on GEO, a unique and persistent identifying number is given to that dataset.

# Mass spectrometry

**5a. How and when will data be shared? Are there possible restrictions to data sharing or embargo reasons?**

This dataset will be made publicly available at the time of manuscript publication:

* analyzed data will be made publicly available in the publications. All manuscripts will be published open access, under a CC-BY-NC license.

* Mass spectrometry data (`.raw` or `.msf` files) associated with the publication will be made publicly available in PRIDE, a well-known repository for mass spectrometry data, and will be given a PRIDE reference number that will be linked in the publication. PRIDE includes extensive experimental metadata with files.

During the project, the dataset will also be shared outside of the immediate team on a case-to-case basis (i.e. with colleagues or collaborators). My data sharing policy and links to all publications will be published on my laboratory website.

**5b. How will data for preservation be selected, and where data will be preserved long-term (for example a data repository or archive)?**

All conclusive and usable data will be stored during the maximum time allowed by the Institut Pasteur storage allowance capacity, the Institut Curie myProMS data management and analysis software, and PRIDE repository. Data will also be stored in external hard disks in the laboratory.

**5c. What methods or software tools are needed to access and use data?**

As data will be stored in `.raw` and `.msf` formats. There are a number of freely accessible mass spectrometry analysis softwares, but we use a software, MyProMS (https://github.com/bioinfo-pf-curie/myproms) that was created by the Institut Curie Laboratoire de Spéctrométrie de Masse Protéomique.

**5d. How will the application of a unique and persistent identifier (such as a Digital Object Identifier (DOI)) to each data set be ensured?**

When published datasets are deposited on PRIDE, a unique and persistent identifying number is given to that dataset, which will
be referenced in the associated publication.

# 6. Data management responsibilities and resources

**6a. Who (for example role, position, and institution) will be responsible for data management (i.e. the data steward)?**

The project coordinator, Jessica Bryant (chargée de recherche), will be responsible for data management.

**6b. What resources (for example financial and time) will be dedicated to data management and ensuring that data will be FAIR (Findable, Accessible, Interoperable, Re-usable)?**

Since all data will be stored on internal Institut Pasteur servers or free public repositories, the only financial resources required for ensuring that data will be FAIR are the cost for external hard-drives and the publication costs for open access journals. The project coordinator, Jessica Bryant, will devote whatever amount of time is required to ensure the policies set forth in this DMP. She will also ensure that all partners and collaborators in the project are aware of and follow the DMP.