BICF ChIPseq is a bioinformatics best-practice analysis pipeline used for ChIP-seq (chromatin immunoprecipitation sequencing) data analysis at BICF at UT Southwestern Department of Bioinformatics.
The pipeline uses Nextflow, a bioinformatics workflow tool. It pre-processes raw data from FastQ inputs, aligns the reads and performs extensive quality-control on the results.
This pipeline is primarily used with a SLURM cluster on the BioHPC Cluster. However, the pipeline should be able to run on any system that supports Nextflow.
Additionally, the pipeline is designed to work with Astrocyte Workflow System using a simple web interface.
Current version of the software and issue reports are at https://git.biohpc.swmed.edu/BICF/Astrocyte/chipseq_analysis
To download the current version of the software
$ git clone [email protected]:BICF/Astrocyte/chipseq_analysis.git- You will need the full path to the files for the Bash Scipt
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The Design file is a tab-delimited file with 8 columns for Single-End and 9 columns for Paired-End. Letter, numbers, and underlines can be used in the names. However, the names can only begin with a letter. Columns must be as follows:
- sample_id a short, unique, and concise name used to label output files; will be used as a control_id if it is the control sample
- experiment_id biosample_treatment_factor; same name given for all replicates of treatment. Will be used for the consensus header.
- biosample symbol for tissue type or cell line
- factor symbol for antibody target
- treatment symbol of treatment applied
- replicate a number, usually from 1-3 (i.e. 1)
- control_id sample_id name that is the control for this sample
- fastq_read1 name of fastq file 1 for SE or PC data
- fastq_read2 name of fastq file 2 for PE data
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See HERE for an example design file, paired-end
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See HERE for an example design file, single-end
- You will need to create a bash script to run the CHIPseq pipeline on BioHPC
- This pipeline has been optimized for the correct partition
- See HERE for an example bash script
- The parameters that must be specified are:
- --reads '/path/to/files/name.fastq.gz'
- --designFile '/path/to/file/design.txt',
- --genome 'GRCm38', 'GRCh38', or 'GRCh37' (if you need to use another genome contact the BICF)
- --pairedEnd 'true' or 'false' (where 'true' is PE and 'false' is SE; default 'false')
- --outDir (optional) path and folder name of the output data, example: /home2/s000000/Desktop/Chipseq_output (if not specficied will be under workflow/output/)
- There are 11 steps to the pipeline
- Check input files
- Trim adaptors TrimGalore!
- Aligned trimmed reads with bwa, and sorts/converts to bam with samtools
- Mark duplicates with Sambamba, and filter reads with samtools
- Quality metrics with deep tools
- Calculate cross-correlation using PhantomPeakQualTools
- Call peaks with MACS
- Calculate consensus peaks
- Annotate all peaks using ChipSeeker
- Calculate Differential Binding Activity with DiffBind (If more than 1 rep in more than 1 experiment)
- Use MEME-ChIP to find motifs in original peaks
See FLOWCHART
| Folder | File | Description |
|---|---|---|
| design | N/A | Inputs used for analysis; can ignore |
| trimReads | *_trimming_report.txt | report detailing how many reads were trimmed |
| trimReads | *_trimmed.fq.gz | trimmed fastq files used for analysis |
| alignReads | *.srt.bam.flagstat.qc | QC metrics from the mapping process |
| alignReads | *.srt.bam | sorted bam file |
| filterReads | *.dup.qc | QC metrics of find duplicate reads (sambamba) |
| filterReads | *.filt.nodup.bam | filtered bam file with duplicate reads removed |
| filterReads | *.filt.nodup.bam.bai | indexed filtered bam file |
| filterReads | *.filt.nodup.flagstat.qc | QC metrics of filtered bam file (mapping stats, samtools) |
| filterReads | *.filt.nodup.pbc.qc | QC metrics of library complexity |
| convertReads | *.filt.nodup.bedse.gz | bed alignment in BEDPE format |
| convertReads | *.filt.nodup.tagAlign.gz | bed alignent in BEDPE format, same as bedse unless samples are paired-end |
| multiqcReport | multiqc_report.html | Quality control report of NRF, PBC1, PBC2, NSC, and RSC. Also contains software versions and references to cite. |
| experimentQC | coverage.pdf | plot to assess the sequencing depth of a given sample |
| experimentQC | *_fingerprint.pdf | plot to determine if the antibody-treatment enriched sufficiently |
| experimentQC | heatmeap_SpearmanCorr.pdf | plot of Spearman correlation between samples |
| experimentQC | heatmeap_PearsonCorr.pdf | plot of Pearson correlation between samples |
| experimentQC | sample_mbs.npz | array of multiple BAM summaries |
| crossReads | *.cc.plot.pdf | Plot of cross-correlation to assess signal-to-noise ratios |
| crossReads | *.cc.qc | cross-correlation metrics. File HEADER |
| callPeaksMACS | pooled/*pooled.fc_signal.bw | bigwig data file; raw fold enrichment of sample/control |
| callPeaksMACS | pooled/*pooled_peaks.xls | Excel file of peaks |
| callPeaksMACS | pooled/*.pvalue_signal.bw | bigwig data file; sample/control signal adjusted for pvalue significance |
| callPeaksMACS | pooled/*_pooled.narrowPeak | peaks file; see HERE for ENCODE narrowPeak header format |
| consensusPeaks | *.rejected.narrowPeak | peaks not supported by multiple testing (replicates and pseudo-replicates) |
| consensusPeaks | *.replicated.narrowPeak | peaks supported by multiple testing (replicates and pseudo-replicates) |
| peakAnnotation | *.chipseeker_annotation.tsv | annotated narrowPeaks file |
| peakAnnotation | *.chipseeker_pie.pdf | pie graph of where narrow annotated peaks occur |
| peakAnnotation | *.chipseeker_upsetplot.pdf | upsetplot showing the count of overlaps of the genes with different annotated location |
| motifSearch | *_memechip/index.html | interactive HTML link of MEME output |
| motifSearch | sorted-*.replicated.narrowPeak | Top 600 peaks sorted by p-value; input for motifSearch |
| motifSearch | *_memechip/combined.meme | MEME identified motifs |
| diffPeaks | heatmap.pdf | Use only for replicated samples; heatmap of relationship of peak location and peak intensity |
| diffPeaks | normcount_peaksets.txt | Use only for replicated samples; peak set values of each sample |
| diffPeaks | pca.pdf | Use only for replicated samples; PCA of peak location and peak intensity |
| diffPeaks | *_diffbind.bed | Use only for replicated samples; bed file of peak locations between replicates |
| diffPeaks | *_diffbind.csv | Use only for replicated samples; CSV file of peaks between replicates |
| plotProfile | plotProfile.png | Plot profile of the TSS region |
| plotProfile | computeMatrix.gz | Compute Matrix from deeptools to create custom plots other than plotProfile |
- These are the list of files that should be reviewed before continuing on with the CHIPseq experiment. If your experiment fails any of these metrics, you should pause and re-evaluate whether the data should remain in the study.
- multiqcReport/multiqc_report.html: follow the ChiP-seq standards HERE;
- experimentQC/*_fingerprint.pdf: make sure the plots information is correct for your antibody/input. See HERE for more details.
- crossReads/*cc.plot.pdf: make sure your sample data has the correct signal intensity and location. See HERE for more details.
- crossReads/*.cc.qc: Column 9 (NSC) should be > 1.1 for experiment and < 1.1 for input. Column 10 (RSC) should be > 0.8 for experiment and < 0.8 for input. See HERE for more details.
- experimentQC/coverage.pdf, experimentQC/heatmeap_SpearmanCorr.pdf, experimentQC/heatmeap_PearsonCorr.pdf: See HERE for more details.
If you find an error, please let the BICF know and we will add it here.
Please cite individual programs and versions used HERE, and the pipeline doi:10.5281/zenodo.2648844. Please cite in publications: Pipeline was developed by BICF from funding provided by Cancer Prevention and Research Institute of Texas (RP150596).
- python/3.6.1-2-anaconda website citation
- trimgalore/0.4.1 website citation
- cutadapt/1.9.1 website citation
- bwa/intel/0.7.12 website citation
- samtools/1.6 website citation
- sambamba/0.6.6 website citation
- bedtools/2.26.0 website citation
- deeptools/2.5.0.1 website citation
- phantompeakqualtools/1.2 website citation
- macs/2.1.0-20151222 website citation
- UCSC_userApps/v317 website citation
- R/3.4.1 website citation
- SPP/1.14
- meme/4.11.1-gcc-openmpi website citation
- ChIPseeker website citation
- DiffBind website citation
This example worklow is derived from original scripts kindly contributed by the Bioinformatic Core Facility (BICF), in the Department of Bioinformatics.