RESTORBIOME2

The aim of this project is to study the transcriptional response of bacteria to complex sugars added to the culture medium. The experiments are carried out on pure culture of microorganisms whose genomes are known.

closed

collaboration

Authors

Affiliation

Olivier Rué

Migale bioinformatics facility

Christelle Hennequet-Antier

Migale bioinformatics facility

Published

May 2, 2023

Modified

February 19, 2025

Note

This document is a report of the analyses performed. You will find all the code used to analyze these data. The version of the tools (maybe in code chunks) and their references are indicated, for questions of reproducibility.

Aim of the project

Patners

Olivier Rué - Migale bioinformatics facility - BioInfomics - INRAE
Christelle Hennequet-Antier - Migale bioinformatics facility - BioInfomics - INRAE
Paul Biscarrat - MICALIS - INRAE
Claire Cherbuy - MICALIS - INRAE

Deliverables

Deliverables agreed at the preliminary meeting (Table 1).

Table 1: Deliverables

	Definition
1	HTML report
2	Archive containing data to be stored

Data management

Important

All data is managed by the migale facility for the duration of the project. Once the project is over, the Migale facility does not keep your data. We will provide you with the raw data and associated metadata that will be deposited on public repositories before the results are used. We can guide you in the submission process. We will then decide which files to keep, knowing that this report will also be provided to you and that the analyses can be replayed if needed.

Raw data

Genomes and mapping data were sent by Paul and deposited on the front server. The genome of E. rectale and its annotation was found on the NCBI website and downloaded. A copy was sent to the abaca server.

Show the code

cd /home/orue/work/PROJECTS/RESTORBIOME2/

Bioinformatics

CAZY annotation

Show the code

sed "s/lcl_NC_008618_1_prot_//g" CAZYMES/BaATCC15703.txt  > CAZYMES/BaATCC15703_refac.txt
sed -i  "s/_1_/\.1_/g" CAZYMES/BaATCC15703_refac.txt
python add_cazymes_to_gff3.py --gff ref/NC_008618_bifido_adolescentis_spikes.gff3 --cazy CAZYMES/BaATCC15703_refac.txt --output NC_008618_bifido_adolescentis_spikes_cazymes.gff3

sed "s/lcl_NZ_AP012325_1_prot_//g" CAZYMES/BcDSM16992.txt > CAZYMES/BcDSM16992_refac.txt
sed -i  "s/_1_/\.1_/g" CAZYMES/BcDSM16992_refac.txt
python add_cazymes_to_gff3.py --gff ref/NZ_AP012325_bifido_catenulatum_spikes.gff3 --cazy CAZYMES/BcDSM16992_refac.txt --output NZ_AP012325_bifido_catenulatum_spikes_cazymes.gff3

sed "s/lcl_CP102263_1_prot_//g" CAZYMES/BuATCC8492.txt > CAZYMES/BuATCC8492_refac.txt
sed -i  "s/_1_/\.1_/g" CAZYMES/BuATCC8492_refac.txt
python add_cazymes_to_gff3.py --gff ref/CP102263_1_Bacteroides_uniformis_spikes.gff3 --cazy CAZYMES/BuATCC8492_refac_ok.txt --output CP102263_1_Bacteroides_uniformis_spikes_cazymes.gff3 --genome BU

sed "s/gene_//g" CAZYMES/ErATCC33656.txt > CAZYMES/ErATCC33656_refac.txt
#sed -i  "s/_1_/\.1_/g" CAZYMES/ErATCC33656_refac.txt
python add_cazymes_to_gff3.py --gff ref/NC_012781_eubacterium_rectale_spikes.gff3 --cazy CAZYMES/ErATCC33656_refac.txt --output NC_012781_eubacterium_rectale_spikes_cazymes.gff3 --genome ER

DBCAN

Show the code

conda activate run_dbcan-3.0.2
run_dbcan ~/work/PROJECTS/RESTORBIOME2/ref/NC_008618_bifido_adolescentis_spikes.fasta prok --out_dir DBCAN_BA --db_dir /db/outils/dbcan/ --tools all --hmm_cpu 16 --eCAMI_jobs 16 --cluster NC_008618_bifido_adolescentis_spikes_cazymes.gff3
python ~/save/SCRIPTS/add_dbcan_to_gff3.py --gff NC_008618_bifido_adolescentis_spikes_cazymes.gff3 --overview DBCAN_BA/overview.txt --gff-prodigal DBCAN_BA/prodigal.gff --output NC_008618_bifido_adolescentis_spikes_cazymes_dbcan.gff3 --cgc-file DBCAN_BA/cgc_standard.out > BA_dbcan.log

run_dbcan ~/work/PROJECTS/RESTORBIOME2/ref/CP102263_1_Bacteroides_uniformis_spikes.fasta prok --out_dir DBCAN_BU --db_dir /db/outils/dbcan/ --tools all --hmm_cpu 16 --eCAMI_jobs 16 --cluster CP102263_1_Bacteroides_uniformis_spikes_cazymes.gff3
python ~/save/SCRIPTS/add_dbcan_to_gff3.py --gff CP102263_1_Bacteroides_uniformis_spikes_cazymes.gff3 --overview DBCAN_BU/overview.txt --gff-prodigal DBCAN_BU/prodigal.gff --output CP102263_1_Bacteroides_uniformis_spikes_cazymes_dbcan.gff3 --cgc-file DBCAN_BU/cgc_standard.out > BU_dbcan.log

run_dbcan ~/work/PROJECTS/RESTORBIOME2/ref/NZ_AP012325_bifido_catenulatum_spikes.fasta prok --out_dir DBCAN_BC --db_dir /db/outils/dbcan/ --tools all --hmm_cpu 16 --eCAMI_jobs 16 --cluster NZ_AP012325_bifido_catenulatum_spikes_cazymes.gff3
python ~/save/SCRIPTS/add_dbcan_to_gff3.py --gff NZ_AP012325_bifido_catenulatum_spikes_cazymes.gff3 --overview DBCAN_BC/overview.txt --gff-prodigal DBCAN_BC/prodigal.gff --output NZ_AP012325_bifido_catenulatum_spikes_cazymes_dbcan.gff3 --cgc-file DBCAN_BC/cgc_standard.out > BC_dbcan.log

run_dbcan ~/work/PROJECTS/RESTORBIOME2/ref/NC_012781_eubacterium_rectale_spikes.fasta prok --out_dir DBCAN_ER --db_dir /db/outils/dbcan/ --tools all --hmm_cpu 16 --eCAMI_jobs 16 --cluster NC_012781_eubacterium_rectale_spikes_cazymes.gff3
python ~/save/SCRIPTS/add_dbcan_to_gff3.py --gff NC_012781_eubacterium_rectale_spikes_cazymes.gff3 --overview DBCAN_ER/overview.txt --gff-prodigal DBCAN_ER/prodigal.gff --output NC_012781_eubacterium_rectale_spikes_cazymes_dbcan.gff3 --cgc-file DBCAN_ER/cgc_standard.out > ER_dbcan.log

PULDB annotation

Show the code

cd /home/orue/work/PROJECTS/RESTORBIOME2

pip install biopython
pip install gff3

# Add pulPred tag to GFF3 for BU only
python add_pul_to_gff3.py --gff CP102263_1_Bacteroides_uniformis_spikes_cazymes_dbcan.gff3 --pul CAZYMES/BuATCC8492_PULs.txt --output CP102263_1_Bacteroides_uniformis_spikes_cazymes_dbcan_pulpred.gff3 --tag pulPred
# Add CC tag to GFF3 for BU only
python add_pul_to_gff3.py --gff CP102263_1_Bacteroides_uniformis_spikes_cazymes_dbcan_pulpred.gff3 --pul CAZYMES/BuATCC8492_CC.txt --output CP102263_1_Bacteroides_uniformis_spikes_cazymes_dbcan_pulpred_cc.gff3 --tag CC

Biostatistics

Setup statistical analyses

We first setup our environment by loading a few packages.

Show the code

library(tidyverse)
library(kableExtra) ## table visualisation
#library(rtracklayer) ## for annotation
library(EnhancedVolcano) ## for volcano plot

We performed differential analyses for each experiment on bacteria:

Bifidobacterium adolescentis (BA)
Bifidobacterium catenulatum (BC)
Bacteroides uniformis (BU)
Eubacterium rectale (ER)

The comparaisons of interest concerned Raffinose (R) treatment versus control treatment as Glucose (G). The lists of differential genes were produced using edgeR R package [1, 2].

Exploratory analyses and normalisation

Reports on normalisation step for each experiment on bacteria

Differential analysis

Reports on differential analysis step for each experiment on bacteria

Excel files with 3 sheets (complete, up, down) for each experiment on bacteria

Enhanced Volcano plot

Volcano plots were performed with EnhancedVolcano R package from Bioconductor project. Each point represents a gene with coordinates x = log2 Fold Change and y = -log10 of adjusted pvalue with Benjamini-Hochberg correction of the contrast tested.

Reports on Volcano visualisation for each experiment on bacteria

Excel files with 3 sheets (complete, pul, cazy) for each experiment on bacteria

References

1. Robinson MD, Oshlack A. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biology. 2010;11:R25. doi:10.1186/gb-2010-11-3-r25.

2. Mc DJ, Chen Y, Smyth GK. Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Res. 2012;40:4288–97.

Reuse

This document will not be accessible without prior agreement of the partners

A work by Migale Bioinformatics Facility
Université Paris-Saclay, INRAE, MaIAGE, 78350, Jouy-en-Josas, France
Université Paris-Saclay, INRAE, BioinfOmics, MIGALE bioinformatics facility, 78350, Jouy-en-Josas, France

--- title: "RESTORBIOME2" search: false navbar: false subtitle: "The aim of this project is to study the transcriptional response of bacteria to complex sugars added to the culture medium. The experiments are carried out on pure culture of microorganisms whose genomes are known." author: - name: Olivier Rué orcid: 0000-0003-1689-0557 email: olivier.rue@inrae.fr affiliations: - name: Migale bioinformatics facility adress: Domaine de Vilvert city: Jouy-en-Josas state: France - name: Christelle Hennequet-Antier orcid: 0000-0001-5836-2803 email: christelle.hennequet-antier@inrae.fr affiliations: - name: Migale bioinformatics facility adress: Domaine de Vilvert city: Jouy-en-Josas state: France date: "2023-05-02" date-modified: today categories: [closed, collaboration] image: "preview.jpg" reader-mode: false fig-cap-location: bottom tbl-cap-location: top license: "This document will not be accessible without prior agreement of the partners" format: html: embed-resources: false toc: true toc-location: right page-layout: article code-overflow: wrap code-fold: true code-tools: true code-summary: "Show the code" --- ```{r setup, include=FALSE} knitr::opts_chunk$set(eval=TRUE, echo =TRUE, cache = FALSE, message = FALSE, warning = FALSE, cache.lazy = FALSE, fig.height = 3.5, fig.width = 10) ``` ```{r load-packages, echo=FALSE, eval=TRUE, cache = FALSE} library(DT) library(scales) library(tidyverse) library(kableExtra) library(phyloseq) library(phyloseq.extended) library(data.table) library(InraeThemes) ``` ```{r datatable global options, echo=FALSE, eval=TRUE} options(DT.options = list(pageLength = 10, scrollX = TRUE, language = list(search = 'Filter:'), dom = 'lftipB', buttons = c('copy', 'csv', 'excel'))) ``` {{< include /resources/_includes/_intro_note.qmd >}} # Aim of the project The aim of this project is to study the transcriptional response of bacteria to complex sugars added to the culture medium. The experiments are carried out on pure culture of microorganisms whose genomes are known. ## Patners * Olivier Rué - Migale bioinformatics facility - BioInfomics - INRAE * Christelle Hennequet-Antier - Migale bioinformatics facility - BioInfomics - INRAE * Paul Biscarrat - MICALIS - INRAE * Claire Cherbuy - MICALIS - INRAE ## Deliverables Deliverables agreed at the preliminary meeting (@tbl-deliverables). |   | Definition | |------|------| | 1 | HTML report | | 2 | Archive containing data to be stored | : Deliverables {#tbl-deliverables} # Data management {{< include /resources/_includes/_data_management.qmd >}} ## Raw data Genomes and mapping data were sent by Paul and deposited on the `front` server. The genome of _E. rectale_ and its annotation was found on the NCBI website and downloaded. A copy was sent to the `abaca` server. ```{bash, eval=F} cd /home/orue/work/PROJECTS/RESTORBIOME2/ ``` # Bioinformatics ## CAZY annotation ```{bash, eval=FALSE} sed "s/lcl_NC_008618_1_prot_//g" CAZYMES/BaATCC15703.txt > CAZYMES/BaATCC15703_refac.txt sed -i "s/_1_/\.1_/g" CAZYMES/BaATCC15703_refac.txt python add_cazymes_to_gff3.py --gff ref/NC_008618_bifido_adolescentis_spikes.gff3 --cazy CAZYMES/BaATCC15703_refac.txt --output NC_008618_bifido_adolescentis_spikes_cazymes.gff3 sed "s/lcl_NZ_AP012325_1_prot_//g" CAZYMES/BcDSM16992.txt > CAZYMES/BcDSM16992_refac.txt sed -i "s/_1_/\.1_/g" CAZYMES/BcDSM16992_refac.txt python add_cazymes_to_gff3.py --gff ref/NZ_AP012325_bifido_catenulatum_spikes.gff3 --cazy CAZYMES/BcDSM16992_refac.txt --output NZ_AP012325_bifido_catenulatum_spikes_cazymes.gff3 sed "s/lcl_CP102263_1_prot_//g" CAZYMES/BuATCC8492.txt > CAZYMES/BuATCC8492_refac.txt sed -i "s/_1_/\.1_/g" CAZYMES/BuATCC8492_refac.txt python add_cazymes_to_gff3.py --gff ref/CP102263_1_Bacteroides_uniformis_spikes.gff3 --cazy CAZYMES/BuATCC8492_refac_ok.txt --output CP102263_1_Bacteroides_uniformis_spikes_cazymes.gff3 --genome BU sed "s/gene_//g" CAZYMES/ErATCC33656.txt > CAZYMES/ErATCC33656_refac.txt #sed -i "s/_1_/\.1_/g" CAZYMES/ErATCC33656_refac.txt python add_cazymes_to_gff3.py --gff ref/NC_012781_eubacterium_rectale_spikes.gff3 --cazy CAZYMES/ErATCC33656_refac.txt --output NC_012781_eubacterium_rectale_spikes_cazymes.gff3 --genome ER ``` ## DBCAN ```{bash, eval=FALSE} conda activate run_dbcan-3.0.2 run_dbcan ~/work/PROJECTS/RESTORBIOME2/ref/NC_008618_bifido_adolescentis_spikes.fasta prok --out_dir DBCAN_BA --db_dir /db/outils/dbcan/ --tools all --hmm_cpu 16 --eCAMI_jobs 16 --cluster NC_008618_bifido_adolescentis_spikes_cazymes.gff3 python ~/save/SCRIPTS/add_dbcan_to_gff3.py --gff NC_008618_bifido_adolescentis_spikes_cazymes.gff3 --overview DBCAN_BA/overview.txt --gff-prodigal DBCAN_BA/prodigal.gff --output NC_008618_bifido_adolescentis_spikes_cazymes_dbcan.gff3 --cgc-file DBCAN_BA/cgc_standard.out > BA_dbcan.log run_dbcan ~/work/PROJECTS/RESTORBIOME2/ref/CP102263_1_Bacteroides_uniformis_spikes.fasta prok --out_dir DBCAN_BU --db_dir /db/outils/dbcan/ --tools all --hmm_cpu 16 --eCAMI_jobs 16 --cluster CP102263_1_Bacteroides_uniformis_spikes_cazymes.gff3 python ~/save/SCRIPTS/add_dbcan_to_gff3.py --gff CP102263_1_Bacteroides_uniformis_spikes_cazymes.gff3 --overview DBCAN_BU/overview.txt --gff-prodigal DBCAN_BU/prodigal.gff --output CP102263_1_Bacteroides_uniformis_spikes_cazymes_dbcan.gff3 --cgc-file DBCAN_BU/cgc_standard.out > BU_dbcan.log run_dbcan ~/work/PROJECTS/RESTORBIOME2/ref/NZ_AP012325_bifido_catenulatum_spikes.fasta prok --out_dir DBCAN_BC --db_dir /db/outils/dbcan/ --tools all --hmm_cpu 16 --eCAMI_jobs 16 --cluster NZ_AP012325_bifido_catenulatum_spikes_cazymes.gff3 python ~/save/SCRIPTS/add_dbcan_to_gff3.py --gff NZ_AP012325_bifido_catenulatum_spikes_cazymes.gff3 --overview DBCAN_BC/overview.txt --gff-prodigal DBCAN_BC/prodigal.gff --output NZ_AP012325_bifido_catenulatum_spikes_cazymes_dbcan.gff3 --cgc-file DBCAN_BC/cgc_standard.out > BC_dbcan.log run_dbcan ~/work/PROJECTS/RESTORBIOME2/ref/NC_012781_eubacterium_rectale_spikes.fasta prok --out_dir DBCAN_ER --db_dir /db/outils/dbcan/ --tools all --hmm_cpu 16 --eCAMI_jobs 16 --cluster NC_012781_eubacterium_rectale_spikes_cazymes.gff3 python ~/save/SCRIPTS/add_dbcan_to_gff3.py --gff NC_012781_eubacterium_rectale_spikes_cazymes.gff3 --overview DBCAN_ER/overview.txt --gff-prodigal DBCAN_ER/prodigal.gff --output NC_012781_eubacterium_rectale_spikes_cazymes_dbcan.gff3 --cgc-file DBCAN_ER/cgc_standard.out > ER_dbcan.log ``` ## PULDB annotation ```{bash, eval=FALSE} cd /home/orue/work/PROJECTS/RESTORBIOME2 pip install biopython pip install gff3 # Add pulPred tag to GFF3 for BU only python add_pul_to_gff3.py --gff CP102263_1_Bacteroides_uniformis_spikes_cazymes_dbcan.gff3 --pul CAZYMES/BuATCC8492_PULs.txt --output CP102263_1_Bacteroides_uniformis_spikes_cazymes_dbcan_pulpred.gff3 --tag pulPred # Add CC tag to GFF3 for BU only python add_pul_to_gff3.py --gff CP102263_1_Bacteroides_uniformis_spikes_cazymes_dbcan_pulpred.gff3 --pul CAZYMES/BuATCC8492_CC.txt --output CP102263_1_Bacteroides_uniformis_spikes_cazymes_dbcan_pulpred_cc.gff3 --tag CC ``` # Biostatistics # Setup statistical analyses We first setup our environment by loading a few packages. ```{r} #| label: packages library(tidyverse) library(kableExtra) ## table visualisation #library(rtracklayer) ## for annotation library(EnhancedVolcano) ## for volcano plot ``` We performed differential analyses for each experiment on bacteria: * *Bifidobacterium adolescentis* (BA) * *Bifidobacterium catenulatum* (BC) * *Bacteroides uniformis* (BU) * *Eubacterium rectale* (ER) The comparaisons of interest concerned Raffinose (R) treatment versus control treatment as Glucose (G). The lists of differential genes were produced using **edgeR** {{< iconify mdi tools >}} R package [@Robinson2010; @McCarthy2012-ij]. # Exploratory analyses and normalisation ```{r} #| label: loopNorm #| include: false # Read counts and construct experimental design # Normalized data # Useful graphics # Create SummarizedExpriment (SE) objects # containing counts, normalized counts experiment <- c("BA", "BC", "BU", "ER") for (i in 1:length(experiment)){ script_name <- paste("1_Explo_Norm_", experiment[i],".qmd", sep='') file.copy(from = "script/1_Explo_Norm_template.qmd", to = file.path("html",script_name), overwrite = TRUE) quarto::quarto_render( input = file.path("html",script_name), execute_params = list( my.interest = experiment[i] ), execute_dir = "./html/", cache_refresh = TRUE, use_freezer = FALSE ) } ``` Reports on normalisation step for each experiment on bacteria * [BA](./html/1_Explo_Norm_BA.html) * [BC](./html/1_Explo_Norm_BC.html) * [BU](./html/1_Explo_Norm_BU.html) * [ER](./html/1_Explo_Norm_ER.html) # Differential analysis ```{r} #| label: loopDE #| include: false # Differential analysis experiment <- c("BA", "BC", "BU", "ER") for (i in 1:length(experiment)){ script_name <- paste("2_Differential_Analysis_", experiment[i],".qmd", sep='') file.copy(from = "script/2_Differential_Analysis_template.qmd", to = file.path("html",script_name), overwrite = TRUE) quarto::quarto_render( input = file.path("html",script_name), execute_params = list( my.interest = experiment[i] ), execute_dir = "./html/", cache_refresh = TRUE, use_freezer = FALSE ) } ``` Reports on differential analysis step for each experiment on bacteria * [BA](./html/2_Differential_Analysis_BA.html) * [BC](./html/2_Differential_Analysis_BC.html) * [BU](./html/2_Differential_Analysis_BU.html) * [ER](./html/2_Differential_Analysis_ER.html) Excel files with 3 sheets (complete, up, down) for each experiment on bacteria * [BA](./html/BA_RvsG.xlsx) * [BC](./html/BC_RvsG.xlsx) * [BU](./html/BU_RvsG.xlsx) * [ER](./html/ER_RvsG.xlsx) # Enhanced Volcano plot Volcano plots were performed with **EnhancedVolcano** {{< iconify mdi tools >}} R package from Bioconductor project. Each point represents a gene with coordinates x = log2 Fold Change and y = -log10 of adjusted pvalue with Benjamini-Hochberg correction of the contrast tested. ```{r} #| label: loopVolcano #| include: false # load a ExperimentSubset object with expression on DE genes # Using es ExperimentSubset object # containing counts logcpm : for all genes, and subset for DE at each contrast experiment <- c("BA", "BC", "BU", "ER") for (i in 1:length(experiment)){ script_name <- paste("4_Volcano_", experiment[i],".qmd", sep='') file.copy(from = "script/4_Volcano_template.qmd", to = file.path("html",script_name), overwrite = TRUE) quarto::quarto_render( input = file.path("html",script_name), execute_params = list( my.interest = experiment[i] ), execute_dir = "./html/", cache_refresh = TRUE, use_freezer = TRUE ) } ``` Reports on Volcano visualisation for each experiment on bacteria * [BA](./html/4_Volcano_BA.html) * [BC](./html/4_Volcano_BC.html) * [BU](./html/4_Volcano_BU.html) * [ER](./html/4_Volcano_ER.html) Excel files with 3 sheets (complete, pul, cazy) for each experiment on bacteria * [BA](./html/BA_RvsG_cazy.xlsx) * [BC](./html/BC_RvsG_cazy.xlsx) * [BU](./html/BU_RvsG_cazy_pul.xlsx) * [ER](./html/ER_RvsG_cazy.xlsx) # References