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indrops_pipeline.md

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+# inDrops
+
+## Installation
+
+Installer avec conda l'environnement `single_cell_snakemake.yaml` ainsi que l'environnement `single_cell.yaml`
+
+```bash
+conda env create -f single_cell_snakemake.yaml
+```
+
+## YAML file configuration
+
+Aide pour régler les problèmes liés au fichier YAML pour plus de détails voir [la page github correspondante](https://github.com/indrops/indrops).
+
+Indiquer le nom du projet et l'emplacement des fichiers de sortie.
+
+```yaml
+project_name : "SingleCell_ICM"
+project_dir : "/home/adrien.dufour/PROTECT/debug_output/"
+```
+
+```yaml
+cores :
+  default : 3
+  quantify_barcodes : 8
+```
+
+Nombre de coeurs pour les différentes étapes, augmenter le nombre de coeurs de `quantify_barcodes` en cas d'erreur samtools
+
+```bash
+[E::hts_open_format] Failed to open file "scRNA_project/sample1/quant_dir/bcIKBG.genomic.sorted.bam" : Too many open files
+samtools merge: fail to open "scRNA_project/sample1/quant_dir/bcIKBG.genomic.sorted.bam": Too many open files
+```
+
+Adapter le nom des fichiers fasta à la nomenclature de la version désirée (v1, v2, v3).
+
+```yaml
+sequencing_runs :
+  - name : "Run1"
+    version : 'v1'
+    dir : "/home/adrien.dufour/PROTECT/debug_data/"
+    fastq_path : "{library_prefix}_{split_affix}_{read}.fastq.gz"
+    split_affixes : ["S3", "S4"]
+    libraries :
+      - {library_name: "AR005", library_prefix: "AR005"}
+      - {library_name: "AR004", library_prefix: "AR004"}
+```
+
+Indiquer l'emplacement des fichiers d'index en `.annotated`
+
+Indiquer l'emplacement du `/bin/` de l'environnement `single_cell_snakemake.yaml` pour Bowtie, Samtools, Rsem et Java
+
+Indiquer l'emplacement du `/bin/` de l'environnement `single_cell.yaml` pour Python
+
+```yaml
+paths :
+  bowtie_index : "/home/adrien.dufour/PROTECT/DOWNLOAD_DIR/Mus_musculus.GRCm38.85.annotated"
+  bowtie_dir : "/opt/miniconda3/bin/"
+  python_dir : "/opt/miniconda3/envs/indrops/bin/"
+  samtools_dir : "/opt/miniconda3/bin/"
+  rsem_dir : "/opt/miniconda3/bin/"
+  java_dir : "/opt/miniconda3/bin/"
+```
+
+Paramètres additionnels
+
+```yaml
+parameters : # OPTIONAL PARAMETERS
+  umi_quantification_arguments:
+    m : 10 #Ignore reads with more than M alignments, after filtering on distance from transcript end.
+    u : 1 #Ignore counts from UMI that should be split among more than U genes.
+    d : 400 #Maximal distance from transcript end, NOT INCLUDING THE POLYA TAIL
+    split-ambigs: False #If umi is assigned to m genes, add 1/m to each gene's count (instead of 1)
+    min_non_polyA: 0 #Require reads to align to this much non-polyA sequence. (Set to 0 to disable filtering on this parameter.)
+  output_arguments:
+    output_alignment_to_bam: True
+    output_unaligned_reads_to_other_fastq: False
+    output_oversequencing_metrics: True
+    output_umifm_calculation_metrics: True
+    output_quant_metrics: True
+  bowtie_arguments:
+    m : 200
+    n : 1
+    l : 15
+    e : 50
+  trimmomatic_arguments:
+    LEADING: "28"
+    SLIDINGWINDOW: "4:20"
+    MINLEN: "16"
+```
+
+## Snakemake file configuration
+
+Indiquer l'emplacement du fichier d'environnement `single_cell.yaml`
+
+Indiquer l'emplacement du fichier yaml de configuration `project.yaml`
+
+```python
+shell.executable("/bin/bash")
+import itertools
+
+conda: "/home/adrien.dufour/NeuroDev_ADD/Envs/single_cell.yaml"
+configfile: "/home/adrien.dufour/NeuroDev_ADD/SingleCell/indrops-master/project.yaml"
+#shell.prefix("conda activate indrops")
+
+YAMLPATH = "/home/adrien.dufour/NeuroDev_ADD/SingleCell/indrops-master/project.yaml"
+```
+
+Modifier en fonction du naming de vos fichiers
+
+```python
+RUN_LIBRARY = []
+FASTQ = []
+LIBRARY = []
+WORKERS = range(config['cores']['default'])
+WORKER = ['1', '2', '3']
+READS = ['R1', 'R2']
+SPLIT = []
+RUN = []
+
+for each in config['sequencing_runs']:
+    RUN = each['name']
+    dir_lib = each['dir']
+    SPLIT = each['split_affixes']
+    RUN_LIBRARY.append((RUN, each['library_name']))
+    LIBRARY.append(each['library_name'])
+```
+
+## Usage
+
+Ce placer dans le dossier contenant le fichier `indrops.py`
+
+```bash
+snakemake --cores 12 --use-conda --conda-prefix "/opt/miniconda3" --latency-wait 25
+```
+
+Commande pour le cluster de Strasbourg
+
+```bash
+snakemake  \
+--configfile /b/home/inci/mokhtari/SingleCell/strasbourg/indrops-master/project-Copy7.yaml \
+--cluster-config /b/home/inci/mokhtari/SingleCell/Script/cluster_config_sc.yaml \
+--snakefile /b/home/inci/mokhtari/SingleCell/strasbourg/indrops-master/Snakefile_7 \
+--jobs 1 \
+--cluster "sbatch --verbose \
+-A ${ACCOUNT} \
+-p ${PARTITION} \
+--time={cluster.walltime} \
+--mem={cluster.mem_gb}G \
+--cpus-per-task={cluster.cpus} \
+--output={cluster.stdout} \
+--error={cluster.stderr}" \
+--use-conda \
+--conda-prefix "/b/home/inci/mokhtari/.conda/envs/singlecell/bin/" \
+--printshellcmds \
+--latency-wait 60 \
+--keep-going
+```
+
+## Troubleshooting
+
+Si votre environnement de travail ne supporte pas les fichiers fifo modifiez la ligne 1231 du fichier `indrops.py`
+
+```python
+filtered_dir = os.path.dirname("/mnt/ram")
+```