3. Tutorials¶
3.1. Initial setup¶
3.1.1. Gene-regulation library¶
For each study presented here we’re creating a link to the gene-regulation library, previously downloaded in section “Quick start”.
Note: if you’re using a clone of the library, you might want to make a copy of it, in order to ensure consistency for later analyses.
3.1.2. Genome directory¶
We chose to define a permanent location for genome downloads, then create symlinks for study cases.
GENOME_DIR=$HOME/genome
mkdir ${GENOME_DIR}
3.2. ChIP-seq study case in S. cerevisiae¶
3.2.1. Presentation¶
Reference
Preti M, Ribeyre C, Pascali C, Bosio MC et al. The telomere-binding protein Tbf1 demarcates snoRNA gene promoters in Saccharomyces cerevisiae. Mol Cell 2010 May 28;38(4):614-20. PMID: 20513435
Abstract
Small nucleolar RNAs (snoRNAs) play a key role in ribosomal RNA biogenesis, yet factors controlling their expression are unknown. We found that the majority of Saccharomyces snoRNA promoters display an aRCCCTaa sequence motif at the upstream border of a TATA-containing nucleosome-free region. Genome-wide ChIP-seq analysis showed that these motifs are bound by Tbf1, a telomere-binding protein known to recognize mammalian-like T(2)AG(3) repeats at subtelomeric regions. Tbf1 has over 100 additional promoter targets, including several other genes involved in ribosome biogenesis and the TBF1 gene itself. Tbf1 is required for full snoRNA expression, yet it does not influence nucleosome positioning at snoRNA promoters. In contrast, Tbf1 contributes to nucleosome exclusion at non-snoRNA promoters, where it selectively colocalizes with the Tbf1-interacting zinc-finger proteins Vid22 and Ygr071c. Our data show that, besides the ribosomal protein gene regulator Rap1, a second telomere-binding protein also functions as a transcriptional regulator linked to yeast ribosome biogenesis.
Access link
- GEO series: GSE20870
3.2.1.1. Download reference genome & annotations¶
mkdir ${GENOME_DIR}/sacCer2
wget -nc ftp://ftp.ensemblgenomes.org/pub/fungi/release-30/fasta/saccharomyces_cerevisiae/dna/Saccharomyces_cerevisiae.R64-1-1.30.dna.genome.fa.gz -P${GENOME_DIR}/sacCer2
wget -nc ftp://ftp.ensemblgenomes.org/pub/fungi/release-30/gff3/saccharomyces_cerevisiae/Saccharomyces_cerevisiae.R64-1-1.30.gff3.gz -P ${GENOME_DIR}/sacCer2
wget -nc ftp://ftp.ensemblgenomes.org/pub/fungi/release-30/gtf/saccharomyces_cerevisiae/Saccharomyces_cerevisiae.R64-1-1.30.gtf.gz -P ${GENOME_DIR}/sacCer2
gunzip ${GENOME_DIR}/sacCer2/*.gz
3.2.1.2. Setup analysis environment¶
ANALYSIS_DIR=$HOME/ChIP-seq_GSE20870
mkdir -p ${ANALYSIS_DIR}
ln -s ${GENE_REG_PATH} gene-regulation
ln -s ${GENOME_DIR}/sacCer2 genome
CONFIG=${ANALYSIS_DIR}/gene-regulation/examples/ChIP-seq_SE_GSE41187/config.yml
3.2.1.3. Download data¶
mkdir -p ${ANALYSIS_DIR}/data/GSM521934 ${ANALYSIS_DIR}/data/GSM521935
wget --no-clobber ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX%2FSRX021%2FSRX021358/SRR051929/SRR051929.sra -P ${ANALYSIS_DIR}/data/GSM521934
wget --no-clobber ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX%2FSRX021%2FSRX021359/SRR051930/SRR051930.sra -P ${ANALYSIS_DIR}/data/GSM521935
show file arborescence
3.2.2. Workflow ‘import_from_sra’¶
The purpose of this workflow is to convert .sra files to .fastq files. The .sra format (Short Read Archive) is used by the GEO database, but for downstream analyses we need to dispose of fastq-formatted files. insert link to glossary section about file formats
It order to run it, you must have followed sections “Setup analysis environment” and “Download data” for the dataset GSE20870.
3.2.2.1. Workflow execution¶
snakemake -s ${ANALYSIS_DIR}/gene-regulation/scripts/snakefiles/workflows/import_from_sra.wf -p --configfile ${CONFIG}
show file arborescence
3.2.3. Workflow ‘quality_control’¶
This workflow can be run after the workflow ‘import_from_sra’, or directly on properly-organized fastq files (see following section if you dispose of your own data).
The purpose of this workflow is to perform quality check with FastQC (link to website and wiki).
Optionally, trimming can be performed using the tool Sickle (link to website and wiki).
cd ${ANALYSIS_DIR}
show file arborescence
3.2.3.1. Workflow execution¶
snakemake -s ${ANALYSIS_DIR}/gene-regulation/scripts/snakefiles/workflows/quality_control.wf -p --configfile ${CONFIG}
show arborescence and/or FastQC screencaps
3.2.4. Workflow ‘ChIP-seq’¶
- This workflows performs:
- mapping with various algorithms;
- genome coverage in different formats (see glossary);
- peak-calling with various algorithms;
- motifs search with suite RSAT (ref).
It order to run it, you must have followed sections “Setup analysis environment” and “Download data”, and “Download genome and annotation” for the dataset GSE20870.
You must have run at least the workflow “import_from_sra’, and optionally the workflow “quality_control”.
cd ${ANALYSIS_DIR}
3.2.4.1. Workflow execution¶
snakemake -s ${ANALYSIS_DIR}/gene-regulation/scripts/snakefiles/workflows/ChIP-seq.wf -p --configfile ${CONFIG}
add figure
3.3. Genome-scale analysis of Escherichia coli FNR¶
3.3.1. Presentation¶
Description
Note: this dataset should be replaced soon by a smaller one
Reference
Myers KS, Yan H, Ong IM, Chung D et al. Genome-scale analysis of Escherichia coli FNR reveals complex features of transcription factor binding. PLoS Genet 2013 Jun;9(6):e1003565. PMID: 23818864
GEO series
3.3.1.1. Download reference genome & annotations¶
mkdir ${GENOME_DIR}/Ecoli-K12
wget -nc ftp://ftp.ensemblgenomes.org/pub/release-21/bacteria/fasta/bacteria_22_collection/escherichia_coli_str_k_12_substr_mg1655/dna/Escherichia_coli_str_k_12_substr_mg1655.GCA_000005845.1.21.dna.genome.fa.gz -P ${GENOME_DIR}/Ecoli-K12
wget -nc ftp://ftp.ensemblgenomes.org/pub/release-21/bacteria/gff3/bacteria_22_collection/escherichia_coli_str_k_12_substr_mg1655/Escherichia_coli_str_k_12_substr_mg1655.GCA_000005845.1.21.gff3.gz -P ${GENOME_DIR}/Ecoli-K12
wget -nc ftp://ftp.ensemblgenomes.org/pub/release-21/bacteria/gtf/bacteria_22_collection/escherichia_coli_str_k_12_substr_mg1655/Escherichia_coli_str_k_12_substr_mg1655.GCA_000005845.1.21.gtf.gz -P ${GENOME_DIR}/Ecoli-K12
gunzip ${GENOME_DIR}/Ecoli-K12/*.gz
3.3.1.2. Setup analysis environment¶
ANALYSIS_DIR=${HOME}/Integrated_analysis
3.3.2. Workflow ‘ChIP-seq’¶
3.3.2.1. Setup analysis environment¶
ANALYSIS_DIR_CHIP=${ANALYSIS_DIR}/ChIP-seq_GSE41187
mkdir -p ${ANALYSIS_DIR_CHIP}
ln -s ${GENE_REG_PATH} ${ANALYSIS_DIR_CHIP}/gene-regulation
ln -s ${GENOME_DIR} ${ANALYSIS_DIR_CHIP}/genome
CONFIG_CHIP=${ANALYSIS_DIR_CHIP}/gene-regulation/examples/ChIP-seq_SE_GSE41187/config.yml
3.3.2.2. Download ChIP-seq data¶
mkdir -p ${ANALYSIS_DIR_CHIP}/data/GSM1010224 ${ANALYSIS_DIR_CHIP}/data/GSM1010219 ${ANALYSIS_DIR_CHIP}/data/GSM1010220
wget --no-clobber ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX%2FSRX189%2FSRX189778/SRR576938/SRR576938.sra -P ${ANALYSIS_DIR_CHIP}/data/GSM1010224
wget --no-clobber ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX%2FSRX189%2FSRX189773/SRR576933/SRR576933.sra -P ${ANALYSIS_DIR_CHIP}/data/GSM1010219
wget --no-clobber ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX/SRX189/SRX189774/SRR576934/SRR576934.sra -P ${ANALYSIS_DIR_CHIP}/data/GSM1010220
3.3.2.3. Workflow execution¶
snakemake -s ${ANALYSIS_DIR_CHIP}/gene-regulation/scripts/snakefiles/workflows/import_to_fastq.wf -p --configfile ${CONFIG_CHIP}
snakemake -s ${ANALYSIS_DIR_CHIP}/gene-regulation/scripts/snakefiles/workflows/quality_control.wf -p --configfile ${CONFIG_CHIP}
snakemake -s ${ANALYSIS_DIR_CHIP}/gene-regulation/scripts/snakefiles/workflows/ChIP-seq.wf -p --configfile ${CONFIG_CHIP}
3.3.3. Workflow ‘RNA-seq’ DEG¶
3.3.3.1. Setup analysis environment¶
ANALYSIS_DIR_RNA=${ANALYSIS_DIR}/RNA-seq_GSE41190
mkdir ${ANALYSIS_DIR_RNA}
ln -s ${GENE_REG_PATH} ${ANALYSIS_DIR_RNA}/gene-regulation
ln -s ${GENOME_DIR} ${ANALYSIS_DIR_RNA}/genome
CONFIG_RNA=${ANALYSIS_DIR_RNA}/gene-regulation/examples/RNA-seq_PE_GSE41190/config.yml
3.3.3.2. Download RNA-seq data¶
mkdir -p ${ANALYSIS_DIR_RNA}/data/GSM1010244 ${ANALYSIS_DIR_RNA}/data/GSM1010245 ${ANALYSIS_DIR_RNA}/data/GSM1010246 ${ANALYSIS_DIR_RNA}/data/GSM1010247
wget --no-clobber ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX/SRX264/SRX2641374/SRR5344681/SRR5344681.sra -P ${ANALYSIS_DIR_RNA}/data/GSM1010244
wget --no-clobber ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX/SRX264/SRX2641375/SRR5344682/SRR5344682.sra -P ${ANALYSIS_DIR_RNA}/data/GSM1010245
wget --no-clobber ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX/SRX264/SRX2641376/SRR5344683/SRR5344683.sra -P ${ANALYSIS_DIR_RNA}/data/GSM1010246
wget --no-clobber ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByExp/sra/SRX/SRX264/SRX2641377/SRR5344684/SRR5344684.sra -P ${ANALYSIS_DIR_RNA}/data/GSM1010247
3.3.3.3. Workflow execution¶
snakemake -s ${ANALYSIS_DIR_RNA}/gene-regulation/scripts/snakefiles/workflows/import_to_fastq.wf -p --configfile ${CONFIG_RNA}
snakemake -s ${ANALYSIS_DIR_RNA}/gene-regulation/scripts/snakefiles/workflows/quality_control.wf -p --configfile ${CONFIG_RNA}
snakemake -s ${ANALYSIS_DIR_RNA}/gene-regulation/scripts/snakefiles/workflows/RNA-seq_workflow_PE.py -p --configfile ${CONFIG_RNA}
3.3.4. Workflow ‘integrated_ChIP_RNA’¶
todo
3.5. Study case yet to find¶
3.5.1. Workflow orthologs¶
todo after we revise the Glossine dataset analysis