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+# Hands-On: Annotation basics
+
+
+
+## Insstallations
+
+Download data from server :
+
+`wget https://dl.pasteur.fr/fop/HJfzm2Py/ChIP_data.tar`
+
+Untar data:
+
+`tar xvf ChIP_data.tar`
+
+Download reference genomes files from server:
+
+`wget https://dl.pasteur.fr/fop/lroDilwn/ReferenceGenome.tar`
+
+Untar data:
+
+`tar xvf ReferenceGenome.tar`
+
+## get ePeak on your home
+
+* Load modules (ON CLUSTER ONLY)
+
+```
+module load snakemake/6.5.0
+module load python/3.7
+module load singularity
+module load git-lfs/2.13.1
+module load pysam
+```
+
+* Clone workflow:
+
+`git clone https://gitlab.pasteur.fr/hub/ePeak.git`
+
+* Download singularity container:
+
+```
+cd ePeak
+singularity pull --arch amd64 --name epeak.img library://rlegendre/epeak/epeak:1.0
+```
+
+## configure ePeak
+
+Open config/config.yaml and config/design.txt files
+
+* **Design file:** tabulated file of 4 columns.
+
+**Column 1** is the name of the IP file
+
+**Column 2** is the name of the corresponding INPUT file
+
+**Column 3** is the replicate number of IP file
+
+**Column 4** is the replicate number of the corresponding INPUT file
+
+
+```
+IP_NAME INPUT_NAME NB_IP NB_INPUT
+H3K27ac_shCtrl INPUT_shCtrl 1 1
+H3K27ac_shCtrl INPUT_shCtrl 2 1
+H3K27ac_shUbc9 INPUT_shUbc9 1 1
+H3K27ac_shUbc9 INPUT_shUbc9 2 1
+Klf4_shCtrl INPUT_shCtrl 1 1
+Klf4_shCtrl INPUT_shCtrl 2 2
+Klf4_shUbc9 INPUT_shUbc9 1 1
+Klf4_shUbc9 INPUT_shUbc9 2 2
+```
+
+* **Config file:** yaml file containing all tools parameters
+
+This file is divided into _chunks_. Each chunk correspond to one step or one tool.
+
+
+This first chunk provides input information and assigns working directories.
+`input_dir` path to FASTQ files directory.
+`input_mate` mate pair format (i.e. `_R[12]` for *MATE* = R1 or R2) , must match the *MATE* parameter in FASTQ files.
+`input_extension` filename extension format (i.e. `fastq.gz` or `fq.gz`).
+`analysis_dir` path to analysis directory.
+`tmpdir` path to temporary directory (i.e. `/tmp/` or other)
+
+```
+input_dir: ../ChIP_data
+input_mate: '_R[12]'
+input_extension: '.fastq.gz'
+analysis_dir: $HOME #define for each user
+tmpdir: $TMPDIR
+```
+
+
+The design chunk aims to check that the FASTQ files name match the design file information. The `marks`, `conditions` and `replicates` parameters must respectively match the *MARK*, *COND* and *REP* parameters of the FASTQ files name and the design file.
+For spike-in data, set `spike` on "True" and provide the spike-in genome FASTA file path through the `spike_genome_file` parameter.
+
+```
+design:
+ design_file: config/design.txt
+ marks: H3K27ac, Klf4
+ condition: shCtrl, shUbc9
+ replicates: Rep
+ spike: false
+ spike_genome_file: genome/dmel9.fa
+```
+
+
+This genome chunk provides information about reference genome - directory, name of the index and path to fasta file.
+
+```
+genome:
+ index: yes
+ genome_directory: genome/
+ name: mm10
+ fasta_file: genome/mm10_chr1.fa
+```
+
+The fastqc chunk provides quality control checking of fastq files.
+
+```
+fastqc:
+ options: ''
+ threads: 4
+```
+
+The adapters chunk is relative to quality trimming and adapter removal with cutadapt. A list of common adapters is provided under config directory and give to cutadapt (adapter_list). Then, different parameters are tuned to match precisely with the data.
+
+
+```
+adapters:
+ remove: yes
+ adapter_list: file:config/adapt.fa
+ m: 25
+ mode: a
+ options: -O 6 --trim-n --max-n 1
+ quality: 30
+ threads: 4
+```
+
+
+The bowtie2_mapping chunk is relative to the reads mapping against genome file (provided by the genome chunk)
+
+```
+bowtie2_mapping:
+ options: "--very-sensitive --no-unal"
+ threads: 4
+```
+
+
+The mark duplicates chunk allows to mark PCR duplicate in BAM files. For ChIPseq data, IP and INPUT need to be deduplicated, so the dedup_IP parameter is set to True.
+
+
+```
+mark_duplicates:
+ do: yes
+ dedup_IP: 'True'
+ threads: 4
+```
+
+The remove_biasedRegions chunk is relative to remove biased genomic regions (previously named blacklisted regions)
+
+```
+remove_biasedRegions:
+ do: yes
+ bed_file: genome/mm10.blacklist.bed
+ threads: 1
+```
+
+To produce metaregion profiles, coverages from each samples need to be producted.
+
+See https://deeptools.readthedocs.io/en/latest/content/feature/effectiveGenomeSize.html
+
+```
+bamCoverage:
+ do: yes
+ options: "--binSize 10 --effectiveGenomeSize 2652783500 --normalizeUsing RPGC"
+ spike-in: no
+ threads: 4
+```
+
+Set yes to geneBody chunk to produce metaregion profiles. This step need a gene model file in bed format.
+
+```
+geneBody:
+ do: yes
+ regionsFileName: genome/mm10_chr1_RefSeq.bed
+ threads: 4
+```
+
+Set all following chunks 'do' to 'no' for now.
+
+
+## run ePeak
+
+
+Test your configuration by performing a dry-run via:
+
+`snakemake --use-singularity -n --cores 1`
+
+Execute the workflow locally using $N cores via:
+
+```
+export PICARD_TOOLS_JAVA_OPTS="-Xmx8G"
+N=8
+snakemake --use-singularity --singularity-args "-B '/home/'" --cores $N
+```
+
+
+Run it specifically on Slurm cluster:
+
+`sbatch snakemake --use-singularity --singularity-args "-B '$HOME'" --cluster-config config/cluster_config.json --cluster "sbatch --mem={cluster.ram} --cpus-per-task={threads} " -j 200 --nolock --cores $SLURM_JOB_CPUS_PER_NODE`
+
+
+## analyse QC reports
+
+### Look at MultiQC report
+
+- General statistics
+
+<img src="images/Multiqc_mainStats.png" width="1000" align="center" >
+
+- Mapping with bowtie2
+
+<img src="images/bowtie2_se_plot.png" width="700" align="center" >
+
+- Deduplication with MarkDuplicates
+
+<img src="images/picard_deduplication.png" width="700" align="center" >
+
+- Fingerplot
+
+<img src="images/deeptools_fingerprint_plot.png" width="700" align="center" >
+
+
+### Look at 05-QC directory
+
+- Cross correlation
+
+ <img src="images/H3K27ac_shCtrl_ppqt.png" width="700" align="center" > <img src="images/Klf4_shCtrl_ppqt.png" width="700" align="center" >
+
+- GeneBody plot/heatmap
+
+<img src="images/geneBodyplot.png" width="700" align="center" >
+
+
+
+
+Would you proceed to the analysis ? justify why