# BICF ChIP-seq Analysis Workflow

## Introduction
**ChIP-seq Analysis** is a bioinformatics best-practice analysis pipeline used for chromatin immunoprecipitation (ChIP-seq) data analysis.

The pipeline uses [Nextflow](https://www.nextflow.io), a bioinformatics workflow tool. It pre-processes raw data from FastQ inputs, aligns the reads and performs extensive quality-control on the results.

Report issues to the Bioinformatic Core Facility [BICF](mailto:BICF@UTSouthwestern.edu)

### Pipeline Steps
  + There are 11 steps to the pipeline
    1. Check input files
    2. Trim adaptors TrimGalore!
    3. Aligned trimmed reads with bwa, and sorts/converts to bam with samtools
    4. Mark duplicates with Sambamba, and filter reads with samtools
    5. Quality metrics with deep tools
    6. Calculate cross-correlation using PhantomPeakQualTools
    7. Call peaks with MACS
    8. Calculate consensus peaks
    9. Annotate all peaks using ChipSeeker
    10. Calculate Differential Binding Activity with DiffBind (If more than 1 rep in more than 1 experiment)
    11. Use MEME-ChIP to find motifs in original peaks


## Workflow Parameters
    1. One or more input FASTQ files from a ChIP-seq expereiment and a design file with the link bewetwen the same file name and sample id (required) - Choose all ChIP-seq fastq files for analysis.
    2. In single-end sequencing, the sequencer reads a fragment from only one end to the other, generating the sequence of base pairs. In paired-end reading it starts at one read, finishes this direction at the specified read length, and then starts another round of reading from the opposite end of the fragment. (Paired-end: True, Single-end: False) (required)
    3. A design file listing sample id, fastq files, corresponding control id and additional information about the sample.
    genome - Choose a genomic reference (genome).
    4. Reference species and genome used for alignment and subsequent analysis. (required)
    5. Run differential peak analysis (required). Must have at least 2 replicates per experiment and at least 2 experiments.
    6. Run motif calling (required). Top 600 peaks sorted by p-value.
    7. Ensure configuration for astrocyte. (required; always true)

## Design file

  + 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:
      1. 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
      2. experiment_id    biosample_treatment_factor; same name given for all replicates of treatment. Will be used for the consensus header.
      3. biosample          symbol for tissue type or cell line
      4. factor                 symbol for antibody target
      5. treatment           symbol of treatment applied
      6. replicate             a number, usually from 1-3 (i.e. 1)
      7. control_id          sample_id name that is the control for this sample
      8. fastq_read1        name of fastq file 1 for SE or PC data
      9. fastq_read2        name of fastq file 2 for PE data


  + See [HERE](https://git.biohpc.swmed.edu/BICF/Astrocyte/chipseq_analysis/blob/master/test_data/design_ENCSR729LGA_PE.txt) for an example design file, paired-end
  + See [HERE](https://git.biohpc.swmed.edu/BICF/Astrocyte/chipseq_analysis/blob/master/test_data/design_ENCSR238SGC_SE.txt) for an example design file, single-end

## Output Files

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](https://git.biohpc.swmed.edu/BICF/Astrocyte/chipseq_analysis/blob/master/docs/xcor_header.txt)
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](https://genome.ucsc.edu/FAQ/FAQformat.html#format12) 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

## Common Quality Control Metrics
  + 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.
    1. multiqcReport/multiqc_report.html: follow the ChiP-seq standards [HERE](https://www.encodeproject.org/chip-seq/);
    2. experimentQC/*_fingerprint.pdf: make sure the plots information is correct for your antibody/input. See [HERE](https://deeptools.readthedocs.io/en/develop/content/tools/plotFingerprint.html) for more details.
    3. crossReads/*cc.plot.pdf: make sure your sample data has the correct signal intensity and location.  See [HERE](https://git.biohpc.swmed.edu/BICF/Astrocyte/chipseq_analysis/blob/master/docs/phantompeaks.md) for more details.
    4. 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](https://genome.ucsc.edu/encode/qualityMetrics.html) for more details.
    5. experimentQC/coverage.pdf, experimentQC/heatmeap_SpearmanCorr.pdf, experimentQC/heatmeap_PearsonCorr.pdf: See [HERE](https://deeptools.readthedocs.io/en/develop/content/list_of_tools.html) for more details.



### Credits
This example worklow is derived from original scripts kindly contributed by the Bioinformatic Core Facility ([BICF](https://www.utsouthwestern.edu/labs/bioinformatics/)), in the [Department of Bioinformatics](https://www.utsouthwestern.edu/departments/bioinformatics/).

Please cite in publications: Pipeline was developed by BICF from funding provided by Cancer Prevention and Research Institute of Texas (RP150596).

### References

  + python/3.6.1-2-anaconda [website](https://www.anaconda.com/download/#linux) [citation](https://git.biohpc.swmed.edu/BICF/Astrocyte/chipseq_analysis/blob/master/docs/references.md)
  + trimgalore/0.4.1 [website](https://github.com/FelixKrueger/TrimGalore) [citation](https://git.biohpc.swmed.edu/BICF/Astrocyte/chipseq_analysis/blob/master/docs/references.md)
  + cutadapt/1.9.1 [website](https://cutadapt.readthedocs.io/en/stable/index.html) [citation](https://git.biohpc.swmed.edu/BICF/Astrocyte/chipseq_analysis/blob/master/docs/references.md)
  + bwa/intel/0.7.12 [website](http://bio-bwa.sourceforge.net/) [citation](https://git.biohpc.swmed.edu/BICF/Astrocyte/chipseq_analysis/blob/master/docs/references.md)
  + samtools/1.6 [website](http://samtools.sourceforge.net/) [citation](https://git.biohpc.swmed.edu/BICF/Astrocyte/chipseq_analysis/blob/master/docs/references.md)
  + sambamba/0.6.6 [website](http://lomereiter.github.io/sambamba/) [citation](https://git.biohpc.swmed.edu/BICF/Astrocyte/chipseq_analysis/blob/master/docs/references.md)
  + bedtools/2.26.0 [website](https://bedtools.readthedocs.io/en/latest/) [citation](https://git.biohpc.swmed.edu/BICF/Astrocyte/chipseq_analysis/blob/master/docs/references.md)
  + deeptools/2.5.0.1 [website](https://deeptools.readthedocs.io/en/develop/) [citation](https://git.biohpc.swmed.edu/BICF/Astrocyte/chipseq_analysis/blob/master/docs/references.md)
  + phantompeakqualtools/1.2 [website](https://github.com/kundajelab/phantompeakqualtools) [citation](https://git.biohpc.swmed.edu/BICF/Astrocyte/chipseq_analysis/blob/master/docs/references.md)
  + macs/2.1.0-20151222 [website](http://liulab.dfci.harvard.edu/MACS/) [citation](https://git.biohpc.swmed.edu/BICF/Astrocyte/chipseq_analysis/blob/master/docs/references.md)
  + UCSC_userApps/v317 [website](https://genome.ucsc.edu/util.html) [citation](https://git.biohpc.swmed.edu/BICF/Astrocyte/chipseq_analysis/blob/master/docs/references.md)
  + R/3.3.2-gccmkl [website](https://www.r-project.org/) [citation](https://git.biohpc.swmed.edu/BICF/Astrocyte/chipseq_analysis/blob/master/docs/references.md)
  + meme/4.11.1-gcc-openmpi [website](http://meme-suite.org/doc/install.html?man_type=web) [citation](https://git.biohpc.swmed.edu/BICF/Astrocyte/chipseq_analysis/blob/master/docs/references.md)
  + ChIPseeker [website](https://bioconductor.org/packages/release/bioc/html/ChIPseeker.html) [citation](https://git.biohpc.swmed.edu/BICF/Astrocyte/chipseq_analysis/blob/master/docs/references.md)
  + DiffBind [website](https://bioconductor.org/packages/release/bioc/html/DiffBind.html) [citation](https://git.biohpc.swmed.edu/BICF/Astrocyte/chipseq_analysis/blob/master/docs/references.md)
  + MultiQC [website](https://multiqc.info/) [citation](https://git.biohpc.swmed.edu/BICF/Astrocyte/chipseq_analysis/blob/master/docs/references.md)
  + BICFChip-seqAnalysisWorkflow [website](https://git.biohpc.swmed.edu/BICF/Astrocyte/chipseq_analysis) [citation](https://git.biohpc.swmed.edu/BICF/Astrocyte/chipseq_analysis/blob/master/docs/references.md)
  + Nextflow [website](https://www.nextflow.io/) [citation](https://git.biohpc.swmed.edu/BICF/Astrocyte/chipseq_analysis/blob/master/docs/references.md)