diff --git a/README.md b/README.md
index e80b3c7c1fa5c7c7088947485e4fbb77d92e401c..1b14f03866caea98197ec45eca3bd44237436f16 100644
--- a/README.md
+++ b/README.md
@@ -35,7 +35,7 @@ $ git clone git@git.biohpc.swmed.edu:BICF/Astrocyte/chipseq_analysis.git
##### 2) 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
+ 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
@@ -62,16 +62,16 @@ $ git clone git@git.biohpc.swmed.edu:BICF/Astrocyte/chipseq_analysis.git
## Pipeline
+ There are 11 steps to the pipeline
1. Check input files
- 2. Trim raw reads with trim galore
+ 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
+ 4. Mark duplicates with Sambamba, and filter reads with samtools
5. Quality metrics with deep tools
- 6. Calculate cross-correlation using phantompeaktools
+ 6. Calculate cross-correlation using PhantomPeakQualTools
7. Call peaks with MACS
8. Calculate consensus peaks
- 9. Annotate Peaks
- 10. Calculate Differential Binding Activity
- 11. Motif Search 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
See [FLOWCHART](docs/flowchart.pdf)
@@ -90,22 +90,21 @@ filterReads | *.filt.nodup.flagstat.qc | QC metrics of filtered bam file (mappin
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 | *.filt.nodup.tagAlign.15.tagAlign.gz.cc.plot.pdf | plot of cross-correlation to assess signal-to-noise ratios
-crossReads | *.filt.nodup.tagAlign.15.tagAlign.gz.cc.qc | cross-correlation metrics. File [HEADER](docs/xcor_header.txt)
-callPeaksMACS | *.fc_signal.bw | bigwig data file; raw fold enrichment of sample/control
-callPeaksMACS | *.pvalue_signal.bw | bigwig data file; sample/control signal adjusted for pvalue significance
-callPeaksMACS | *_peaks.narrowPeak | peaks file; see [HERE](https://genome.ucsc.edu/FAQ/FAQformat.html#format12) for ENCODE narrowPeak header format
-consensusPeaks | design_annotatePeaks.tsv | design file; can ignore
-consensusPeaks | design_diffPeaks.csv | design file; can ignore
+crossReads | *.cc.plot.pdf | Plot of cross-correlation to assess signal-to-noise ratios
+crossReads | *.cc.qc | cross-correlation metrics. File [HEADER](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)
-consensusPeaks | unique_experiments.csv | design file; can ignore
-peakAnnotation | *.chipseeker_annotation.csv | annotated narrowPeaks file
+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
@@ -119,10 +118,11 @@ diffPeaks | *_diffbind.csv | Use only for replicated samples; CSV file of peaks
## 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. filterReads/*.filt.nodup.pbc.qc: follow the ChiP-seq standards [HERE](https://www.encodeproject.org/chip-seq/); NRF>0.9, PBC1>0.9, and PBC2>10
+ 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/*.filt.nodup.tagAlign.15.tagAlign.gz.cc.plot.pdf: make sure your sample data has the correct signal intensity and location. See [HERE](https://hbctraining.github.io/Intro-to-ChIPseq/lessons/06_QC_cross_correlation.html) for more details.
- 4. crossReads/*.filt.nodup.tagAlign.15.tagAlign.gz.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://hbctraining.github.io/Intro-to-ChIPseq/lessons/06_QC_cross_correlation.html) for more details.
+ 3. crossReads/*cc.plot.pdf: make sure your sample data has the correct signal intensity and location. See [HERE](https://hbctraining.github.io/Intro-to-ChIPseq/lessons/06_QC_cross_correlation.html) 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.
## Common Errors
@@ -140,15 +140,19 @@ If you find an error, please let the [BICF](mailto:BICF@UTSouthwestern.edu) know
+ phantompeakqualtools/1.2 [website](https://github.com/kundajelab/phantompeakqualtools) [citation](docs/references.txt)
+ macs/2.1.0-20151222 [website](http://liulab.dfci.harvard.edu/MACS/) [citation](docs/references.txt)
+ UCSC_userApps/v317 [website](https://genome.ucsc.edu/util.html) [citation](docs/references.txt)
- + R/3.3.2-gccmkl [website](https://www.r-project.org/) [citation](docs/references.txt)
+ + R/3.4.1 [website](https://www.r-project.org/) [citation](docs/references.txt)
+ + SPP/1.14
+ meme/4.11.1-gcc-openmpi [website](http://meme-suite.org/doc/install.html?man_type=web) [citation](docs/references.txt)
+ ChIPseeker [website](https://bioconductor.org/packages/release/bioc/html/ChIPseeker.html) [citation](docs/references.txt)
+ DiffBind [website](https://bioconductor.org/packages/release/bioc/html/DiffBind.html) [citation](docs/references.txt)
+
## 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).
+
## Citation
Please cite individual programs and versions used [HERE](docs/references.txt). Also, please look out for our pipeline to be published in the future [HERE](https://zenodo.org/).
diff --git a/docs/index.md b/docs/index.md
index b6c66cec174ba4aeca2216c39f18dda3bfa89010..6d3d40b8dbe200880f01ecc5878ad160b4775b41 100644
--- a/docs/index.md
+++ b/docs/index.md
@@ -5,61 +5,122 @@
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.
-### Pipeline Steps
+Report issues to the Bioinformatic Core Facility [BICF](mailto:BICF@UTSouthwestern.edu)
-1) Trim adaptors TrimGalore!
-2) Align with BWA
-3) Filter reads with Sambamba S
-4) Quality control with DeepTools
-5) Calculate Cross-correlation using SPP and PhantomPeakQualTools
-6) Signal profiling using MACS2
-7) Call consenus peaks
-8) Annotate all peaks using ChipSeeker
-9) Use MEME-ChIP to find motifs in original peaks
-10) Find differential expressed peaks using DiffBind (If more than 1 experiment)
+### 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
- reads - Choose all ChIP-seq fastq files for analysis.
- pairedEnd - Choose True/False if data is paired-end
- design - Choose the file with the experiment design information. TSV format
+ 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 configuraton for astrocyte. (required; always true)
## Design file
- The following columns are necessary, must be named as in template. An design file template can be downloaded [HERE](https://git.biohpc.swmed.edu/bchen4/chipseq_analysis/raw/master/docs/design_example.csv)
-
- SampleID
- The id of the sample. This will be the header in output files, please make sure it is concise
- Tissue
- Tissue of the sample
- Factor
- Factor of the experiment
- Condition
- This is the group that will be used for pairwise differential expression analysis
- Replicate
- Replicate id
- Peaks
- The file name of the peak file for this sample
- bamReads
- The file name of the IP BAM for this sample
- bamControl
- The file name of the control BAM for this sample
- ContorlID
- The id of the control sample
- PeakCaller
- The peak caller used
+ + 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](test_data/design_ENCSR729LGA_PE.txt) for an example design file, paired-end
+ + See [HERE](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](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
+
+## 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://hbctraining.github.io/Intro-to-ChIPseq/lessons/06_QC_cross_correlation.html) 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), Department of Bioinformatics
+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
-* ChipSeeker: http://bioconductor.org/packages/release/bioc/html/ChIPseeker.html
-* DiffBind: http://bioconductor.org/packages/release/bioc/html/DiffBind.html
-* Deeptools: https://deeptools.github.io/
-* MEME-ChIP: http://meme-suite.org/doc/meme-chip.html
+ + python/3.6.1-2-anaconda [website](https://www.anaconda.com/download/#linux) [citation](docs/references.txt)
+ + trimgalore/0.4.1 [website](https://github.com/FelixKrueger/TrimGalore) [citation](docs/references.txt)
+ + cutadapt/1.9.1 [website](https://cutadapt.readthedocs.io/en/stable/index.html) [citation](docs/references.txt)
+ + bwa/intel/0.7.12 [website](http://bio-bwa.sourceforge.net/) [citation](docs/references.txt)
+ + samtools/1.6 [website](http://samtools.sourceforge.net/) [citation](docs/references.txt)
+ + sambamba/0.6.6 [website](http://lomereiter.github.io/sambamba/) [citation](docs/references.txt)
+ + bedtools/2.26.0 [website](https://bedtools.readthedocs.io/en/latest/) [citation](docs/references.txt)
+ + deeptools/2.5.0.1 [website](https://deeptools.readthedocs.io/en/develop/) [citation](docs/references.txt)
+ + phantompeakqualtools/1.2 [website](https://github.com/kundajelab/phantompeakqualtools) [citation](docs/references.txt)
+ + macs/2.1.0-20151222 [website](http://liulab.dfci.harvard.edu/MACS/) [citation](docs/references.txt)
+ + UCSC_userApps/v317 [website](https://genome.ucsc.edu/util.html) [citation](docs/references.txt)
+ + R/3.3.2-gccmkl [website](https://www.r-project.org/) [citation](docs/references.txt)
+ + meme/4.11.1-gcc-openmpi [website](http://meme-suite.org/doc/install.html?man_type=web) [citation](docs/references.txt)
+ + ChIPseeker [website](https://bioconductor.org/packages/release/bioc/html/ChIPseeker.html) [citation](docs/references.txt)
+ + DiffBind [website](https://bioconductor.org/packages/release/bioc/html/DiffBind.html) [citation](docs/references.txt)