Last updated: 2019-03-02

Checks: 6 0

Knit directory: threeprimeseq/analysis/

This reproducible R Markdown analysis was created with workflowr (version 1.2.0). The Report tab describes the reproducibility checks that were applied when the results were created. The Past versions tab lists the development history.

R Markdown file: up-to-date

Great! Since the R Markdown file has been committed to the Git repository, you know the exact version of the code that produced these results.

Environment: empty

Great job! The global environment was empty. Objects defined in the global environment can affect the analysis in your R Markdown file in unknown ways. For reproduciblity it’s best to always run the code in an empty environment.

Seed: set.seed(12345)

The command set.seed(12345) was run prior to running the code in the R Markdown file. Setting a seed ensures that any results that rely on randomness, e.g. subsampling or permutations, are reproducible.

Session information: recorded

Great job! Recording the operating system, R version, and package versions is critical for reproducibility.

Cache: none

Nice! There were no cached chunks for this analysis, so you can be confident that you successfully produced the results during this run.

Repository version: 49f21df

Great! You are using Git for version control. Tracking code development and connecting the code version to the results is critical for reproducibility. The version displayed above was the version of the Git repository at the time these results were generated.

Note that you need to be careful to ensure that all relevant files for the analysis have been committed to Git prior to generating the results (you can use wflow_publish or wflow_git_commit). workflowr only checks the R Markdown file, but you know if there are other scripts or data files that it depends on. Below is the status of the Git repository when the results were generated: 


Ignored files:
    Ignored:    .DS_Store
    Ignored:    .Rhistory
    Ignored:    .Rproj.user/
    Ignored:    data/.DS_Store
    Ignored:    data/perm_QTL_trans_noMP_5percov/
    Ignored:    output/.DS_Store

Untracked files:
    Untracked:  KalistoAbundance18486.txt
    Untracked:  analysis/4suDataIGV.Rmd
    Untracked:  analysis/DirectionapaQTL.Rmd
    Untracked:  analysis/EvaleQTLs.Rmd
    Untracked:  analysis/YL_QTL_test.Rmd
    Untracked:  analysis/groSeqAnalysis.Rmd
    Untracked:  analysis/ncbiRefSeq_sm.sort.mRNA.bed
    Untracked:  analysis/snake.config.notes.Rmd
    Untracked:  analysis/verifyBAM.Rmd
    Untracked:  analysis/verifybam_dubs.Rmd
    Untracked:  code/PeaksToCoverPerReads.py
    Untracked:  code/strober_pc_pve_heatmap_func.R
    Untracked:  data/18486.genecov.txt
    Untracked:  data/APApeaksYL.total.inbrain.bed
    Untracked:  data/AllPeak_counts/
    Untracked:  data/ApaQTLs/
    Untracked:  data/ApaQTLs_otherPhen/
    Untracked:  data/ChromHmmOverlap/
    Untracked:  data/DistTXN2Peak_genelocAnno/
    Untracked:  data/FeatureoverlapPeaks/
    Untracked:  data/GM12878.chromHMM.bed
    Untracked:  data/GM12878.chromHMM.txt
    Untracked:  data/LianoglouLCL/
    Untracked:  data/LocusZoom/
    Untracked:  data/LocusZoom_Unexp/
    Untracked:  data/LocusZoom_proc/
    Untracked:  data/MatchedSnps/
    Untracked:  data/NuclearApaQTLs.txt
    Untracked:  data/PeakCounts/
    Untracked:  data/PeakCounts_noMP_5perc/
    Untracked:  data/PeakCounts_noMP_genelocanno/
    Untracked:  data/PeakUsage/
    Untracked:  data/PeakUsage_noMP/
    Untracked:  data/PeakUsage_noMP_GeneLocAnno/
    Untracked:  data/PeaksUsed/
    Untracked:  data/PeaksUsed_noMP_5percCov/
    Untracked:  data/QTL_overlap/
    Untracked:  data/RNAkalisto/
    Untracked:  data/RefSeq_annotations/
    Untracked:  data/Replicates_usage/
    Untracked:  data/TotalApaQTLs.txt
    Untracked:  data/Totalpeaks_filtered_clean.bed
    Untracked:  data/UnderstandPeaksQC/
    Untracked:  data/WASP_STAT/
    Untracked:  data/YL-SP-18486-T-combined-genecov.txt
    Untracked:  data/YL-SP-18486-T_S9_R1_001-genecov.txt
    Untracked:  data/YL_QTL_test/
    Untracked:  data/apaExamp/
    Untracked:  data/apaExamp_proc/
    Untracked:  data/apaQTL_examp_noMP/
    Untracked:  data/bedgraph_peaks/
    Untracked:  data/bin200.5.T.nuccov.bed
    Untracked:  data/bin200.Anuccov.bed
    Untracked:  data/bin200.nuccov.bed
    Untracked:  data/clean_peaks/
    Untracked:  data/comb_map_stats.csv
    Untracked:  data/comb_map_stats.xlsx
    Untracked:  data/comb_map_stats_39ind.csv
    Untracked:  data/combined_reads_mapped_three_prime_seq.csv
    Untracked:  data/diff_iso_GeneLocAnno/
    Untracked:  data/diff_iso_proc/
    Untracked:  data/diff_iso_trans/
    Untracked:  data/eQTLs_Lietal/
    Untracked:  data/ensemble_to_genename.txt
    Untracked:  data/example_gene_peakQuant/
    Untracked:  data/explainProtVar/
    Untracked:  data/filtPeakOppstrand_cov_noMP_GeneLocAnno_5perc/
    Untracked:  data/filtered_APApeaks_merged_allchrom_refseqTrans.closest2End.bed
    Untracked:  data/filtered_APApeaks_merged_allchrom_refseqTrans.closest2End.noties.bed
    Untracked:  data/first50lines_closest.txt
    Untracked:  data/gencov.test.csv
    Untracked:  data/gencov.test.txt
    Untracked:  data/gencov_zero.test.csv
    Untracked:  data/gencov_zero.test.txt
    Untracked:  data/gene_cov/
    Untracked:  data/joined
    Untracked:  data/leafcutter/
    Untracked:  data/merged_combined_YL-SP-threeprimeseq.bg
    Untracked:  data/molPheno_noMP/
    Untracked:  data/mol_overlap/
    Untracked:  data/mol_pheno/
    Untracked:  data/nom_QTL/
    Untracked:  data/nom_QTL_opp/
    Untracked:  data/nom_QTL_trans/
    Untracked:  data/nuc6up/
    Untracked:  data/nuc_10up/
    Untracked:  data/other_qtls/
    Untracked:  data/pQTL_otherphen/
    Untracked:  data/pacbio_cov/
    Untracked:  data/peakPerRefSeqGene/
    Untracked:  data/peaks4DT/
    Untracked:  data/perm_QTL/
    Untracked:  data/perm_QTL_GeneLocAnno_noMP_5percov/
    Untracked:  data/perm_QTL_GeneLocAnno_noMP_5percov_3UTR/
    Untracked:  data/perm_QTL_diffWindow/
    Untracked:  data/perm_QTL_opp/
    Untracked:  data/perm_QTL_trans/
    Untracked:  data/perm_QTL_trans_filt/
    Untracked:  data/protAndAPAAndExplmRes.Rda
    Untracked:  data/protAndAPAlmRes.Rda
    Untracked:  data/protAndExpressionlmRes.Rda
    Untracked:  data/reads_mapped_three_prime_seq.csv
    Untracked:  data/smash.cov.results.bed
    Untracked:  data/smash.cov.results.csv
    Untracked:  data/smash.cov.results.txt
    Untracked:  data/smash_testregion/
    Untracked:  data/ssFC200.cov.bed
    Untracked:  data/temp.file1
    Untracked:  data/temp.file2
    Untracked:  data/temp.gencov.test.txt
    Untracked:  data/temp.gencov_zero.test.txt
    Untracked:  data/threePrimeSeqMetaData.csv
    Untracked:  data/threePrimeSeqMetaData55Ind.txt
    Untracked:  data/threePrimeSeqMetaData55Ind.xlsx
    Untracked:  data/threePrimeSeqMetaData55Ind_noDup.txt
    Untracked:  data/threePrimeSeqMetaData55Ind_noDup.xlsx
    Untracked:  data/threePrimeSeqMetaData55Ind_noDup_WASPMAP.txt
    Untracked:  data/threePrimeSeqMetaData55Ind_noDup_WASPMAP.xlsx
    Untracked:  output/LZ/
    Untracked:  output/deeptools_plots/
    Untracked:  output/picard/
    Untracked:  output/plots/
    Untracked:  output/qual.fig2.pdf

Unstaged changes:
    Modified:   analysis/28ind.peak.explore.Rmd
    Modified:   analysis/CompareLianoglouData.Rmd
    Modified:   analysis/NewPeakPostMP.Rmd
    Modified:   analysis/apaQTLoverlapGWAS.Rmd
    Modified:   analysis/cleanupdtseq.internalpriming.Rmd
    Modified:   analysis/coloc_apaQTLs_protQTLs.Rmd
    Modified:   analysis/dif.iso.usage.leafcutter.Rmd
    Modified:   analysis/diff_iso_pipeline.Rmd
    Modified:   analysis/explainpQTLs.Rmd
    Modified:   analysis/explore.filters.Rmd
    Modified:   analysis/fixBWChromNames.Rmd
    Modified:   analysis/flash2mash.Rmd
    Modified:   analysis/mispriming_approach.Rmd
    Modified:   analysis/overlapMolQTL.Rmd
    Modified:   analysis/overlapMolQTL.opposite.Rmd
    Modified:   analysis/overlap_qtls.Rmd
    Modified:   analysis/peakOverlap_oppstrand.Rmd
    Modified:   analysis/peakQCPPlots.Rmd
    Modified:   analysis/pheno.leaf.comb.Rmd
    Modified:   analysis/pipeline_55Ind.Rmd
    Modified:   analysis/swarmPlots_QTLs.Rmd
    Modified:   analysis/test.max2.Rmd
    Modified:   analysis/test.smash.Rmd
    Modified:   analysis/understandPeaks.Rmd
    Modified:   analysis/unexplainedeQTL_analysis.Rmd
    Modified:   code/Snakefile

Note that any generated files, e.g. HTML, png, CSS, etc., are not included in this status report because it is ok for generated content to have uncommitted changes.

These are the previous versions of the R Markdown and HTML files. If you’ve configured a remote Git repository (see ?wflow_git_remote), click on the hyperlinks in the table below to view them.

File Version Author Date Message
Rmd 49f21df Briana Mittleman 2019-03-02 modify analysis for len 6 add qtl match
html 901e191 Briana Mittleman 2019-03-02 Build site.
Rmd a96297c Briana Mittleman 2019-03-02 add signal site enrich analysis

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC310884/ (50bp up)

Singal site in peak

I will use homer to look for enriched signals in my signal sites.

Intall homer in my enironment with

conda install wget samtools r-essentials bioconductor-deseq2 bioconductor-edger

I need to get the 50 base pairs upstream of my peaks and the shuffled peaks. This should encompase the signal site. This is similar to the 10 bp upstream script I used to identify evidence of mispriming. I want it to take any peak file and conver the bed to the 50bp uptream.

Upstream50Bases.py

#python  
def main(Fin, Fout):
  outBed=open(Fout, "w")
  chrom_lengths=open("/project2/gilad/briana/genome_anotation_data/chrom_lengths2.sort.bed","r")
  #make a dictionary with chrom lengths
  length_dic={}
  for i in chrom_lengths:
    chrom, start, end = i.split()
    length_dic[str(chrom)]=int(end)  
#write file 
  for ln in open(Fin):
    chrom, start, end, name, score, strand = ln.split()
    chrom=str(chrom)
    if strand=="+":
      start_new=int(start)-50
      if start_new <= 1:
        start_new = 0 
      end_new= int(start)
      if end_new == 0:
        end_new=1
      outBed.write("%s\t%d\t%d\t%s\t%s\t%s\n"%(chrom, start_new, end_new, name, score, strand))
    if strand == "-":
      start_new=int(end)
      end_new=int(end) + 50
      outBed.write("%s\t%d\t%d\t%s\t%s\t%s\n"%(chrom, start_new, end_new, name, score, strand))
  outBed.close()  
if __name__ == "__main__":
    import sys
    inFile = sys.argv[1]
    outFile=sy.argv[2] 
    main(inFile, outFile)

RUn this for both the peak and shuff file with the fixed peak strands:

  • /project2/gilad/briana/threeprimeseq/data/FeatureoverlapPeaks/shuffled_FilterPeaks.sort.bed

  • /project2/gilad/briana/threeprimeseq/data/peaks4DT/APAPeaks_5percCov_fixedStrand.bed

run_get50up.sh

#!/bin/bash

#SBATCH --job-name=run_get50up
#SBATCH --account=pi-yangili1
#SBATCH --time=36:00:00
#SBATCH --output=run_get50upt.out
#SBATCH --error=run_get50up.err
#SBATCH --partition=broadwl
#SBATCH --mem=16G
#SBATCH --mail-type=END

module load Anaconda3
source activate three-prime-env

python Upstream50Bases.py /project2/gilad/briana/threeprimeseq/data/FeatureoverlapPeaks/shuffled_FilterPeaks.sort.bed /project2/gilad/briana/threeprimeseq/data/SignalEnrich/fiftyup_shuffled_FilterPeaks.sort.bed 

python Upstream50Bases.py /project2/gilad/briana/threeprimeseq/data/peaks4DT/APAPeaks_5percCov_fixedStrand.bed /project2/gilad/briana/threeprimeseq/data/SignalEnrich/fiftyup_APAPeaks_5percCov_fixedStrand.bed

Run homer enrichement

-h hypergeometic enrichment

run it in downloaded version for now- will fix when environment solves and this is in anaconda env.

signalSiteEnrich.sh

#!/bin/bash

#SBATCH --job-name=signalSiteEnrich
#SBATCH --account=pi-yangili1
#SBATCH --time=36:00:00
#SBATCH --output=signalSiteEnrich.out
#SBATCH --error=signalSiteEnrich.err
#SBATCH --partition=bigmem2
#SBATCH --mem=100G
#SBATCH --mail-type=END
source deactivate three-prime-env
module unload Anaconda3

findMotifsGenome.pl /project2/gilad/briana/threeprimeseq/data/peaks4DT/APAPeaks_5percCov_fixedStrand.bed /project2/gilad/briana/genome_anotation_data/genome/Homo_sapiens.GRCh37.75.dna_sm.all.fa /project2/gilad/briana/threeprimeseq/data/SignalEnrich/ -size -300,100 -h -bg  /project2/gilad/briana/threeprimeseq/data/FeatureoverlapPeaks/shuffled_FilterPeaks.sort.bed -len 6

Try this with the actual peaks and change the size

Signal site in qtls

/project2/gilad/briana/threeprimeseq/data/ApaQTLs/Nuclear.apaQTLs.sort.bed /project2/gilad/briana/threeprimeseq/data/ApaQTLs/Total.apaQTLs.sort.bed

cat /project2/gilad/briana/threeprimeseq/data/ApaQTLs/Nuclear.apaQTLs.sort.bed /project2/gilad/briana/threeprimeseq/data/ApaQTLs/Total.apaQTLs.sort.bed | sort -k1,1 -k2,2n > /project2/gilad/briana/threeprimeseq/data/ApaQTLs/All.apaQTLs.sort.bed
cat /project2/gilad/briana/threeprimeseq/data/MatchedSnp/Nuclear_matched_snps_sort.bed /project2/gilad/briana/threeprimeseq/data/MatchedSnp/Total_matched_snps_sort.bed  | sort -k1,1 -k2,2n > /project2/gilad/briana/threeprimeseq/data/MatchedSnp/All_matched_snps_sort.bed

signalSiteEnrich_QTLs.sh

#!/bin/bash

#SBATCH --job-name=signalSiteEnrich_QTL
#SBATCH --account=pi-yangili1
#SBATCH --time=36:00:00
#SBATCH --output=signalSiteEnrich_QTL.out
#SBATCH --error=signalSiteEnrich_QTL.err
#SBATCH --partition=bigmem2
#SBATCH --mem=100G
#SBATCH --mail-type=END

#source deactivate three-prime-env
#module unload Anaconda3

findMotifsGenome.pl /project2/gilad/briana/threeprimeseq/data/ApaQTLs/All.apaQTLs.sort.bed /project2/gilad/briana/genome_anotation_data/genome/Homo_sapiens.GRCh37.75.dna_sm.all.fa /project2/gilad/briana/threeprimeseq/data/SignalEnrich_QTL/ -size 50 -h -bg  /project2/gilad/briana/threeprimeseq/data/MatchedSnp/All_matched_snps_sort.bed -len 6

results dont mean much at the moment



sessionInfo()
R version 3.5.1 (2018-07-02)
Platform: x86_64-apple-darwin15.6.0 (64-bit)
Running under: macOS  10.14.1

Matrix products: default
BLAS: /Library/Frameworks/R.framework/Versions/3.5/Resources/lib/libRblas.0.dylib
LAPACK: /Library/Frameworks/R.framework/Versions/3.5/Resources/lib/libRlapack.dylib

locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     

loaded via a namespace (and not attached):
 [1] workflowr_1.2.0 Rcpp_0.12.19    lattice_0.20-35 digest_0.6.17  
 [5] rprojroot_1.3-2 grid_3.5.1      jsonlite_1.6    backports_1.1.2
 [9] git2r_0.24.0    magrittr_1.5    evaluate_0.13   stringi_1.2.4  
[13] fs_1.2.6        whisker_0.3-2   Matrix_1.2-14   reticulate_1.10
[17] rmarkdown_1.11  tools_3.5.1     stringr_1.4.0   glue_1.3.0     
[21] yaml_2.2.0      compiler_3.5.1  htmltools_0.3.6 knitr_1.20