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https://github.com/bartongroup/KF_arabidopsis-GRNA

The scripts bundle included with the manuscript.
https://github.com/bartongroup/KF_arabidopsis-GRNA
Last synced: 3 months ago
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The scripts bundle included with the manuscript.
Host: GitHub
URL: https://github.com/bartongroup/KF_arabidopsis-GRNA
Owner: bartongroup
Created: 2017-11-15T11:53:29.000Z (almost 7 years ago)
Default Branch: master
Last Pushed: 2019-01-28T17:00:27.000Z (almost 6 years ago)
Last Synced: 2024-03-26T23:07:23.858Z (8 months ago)
Language: Perl
Size: 473 KB
Stars: 1
Watchers: 5
Forks: 0
Open Issues: 0
Metadata Files:
- Readme: README.md
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README

        # KF_arabidopsis-GRNA

Scripts and commands for reproducing the analysis in ["How well do RNA-Seq differential gene expression tools perform in a eukaryote with a complex transcriptome?", Froussios, Schurch, Mackinnon, Gierlinski, Duc, Simpson & Barton

doi:10.1101/090753](https://doi.org/10.1101/090753)

# REQUIREMENTS:

How you install these requirements and make them accessible in your PATH is up to you.

1. Sun Grid Engine computing queue manager (some of our scripts internally submit tasks to a computing cluster)

2. R (3.2.2) with: lattice, ColourBrewer, PoissonSeq, edger, limma, deseq, deseq2, degseq, bayseq, ebseq, noiseq, samseq

3. PLplot

4. Perl (5.10.1) with: DRMAAc, PDL, PDL::Graphics::PLplot, DBI, Statistics::R, Devel::Size, Math::CDF, Time::HiRes

5. Python 2.7 with: numpy, scipy, drmaa, pysqlite

6. Python 3 with: pandas

7. STAR (2.5.0)

8. subread (1.6.1)

9. SAMtools (1.7)

Environment variables (assuming Unix/Bash):

	export PROJECTROOT=${HOME}/PROJECTS/AtGRNA

	export PERLROOT=${HOME}/perl5/perlbrew/perls/perl-5.10.1/

	export LOCALEXECROOT=${HOME}/local_installs

	export LD_LIBRARY_PATH=${SGE_ROOT}/lib/`${SGE_ROOT}/util/arch`

	export DRMAA_LIBRARY_PATH=${LD_LIBRARY_PATH}/libdrmaa.sa

	export PYTHONPATH=${PROJECTROOT}/analysis:${PROJECTROOT}/analysis/grnascripts/Modules

	export PERL5LIB=${PROJECTROOT}/analysis/grnascripts/Modules:${PROJECTROOT}/analysis/rnascripts:${PERLROOT}/lib/5.10.1:${PERL5ROOT}/lib/site_perl/5.10.1:${PERL5LIB}

You will likely need to change the paths in the above variables to match your system, especially the first three.

# PROCESSING STEPS

Commands are executed from the base directory of the code distribution. 

## Setup

First, create a project directory and navigate into it. Then clone this repository, get the Araport 11 *Arabidopsis thaliana* annotation and the TAIR10 *A. thaliana* genome assembly, and the raw read files from ArrayExpress.

	mkdir AtGRNA

	cd AtGRNA

	git clone [email protected]:bartongroup/KF_arabidopsis-GRNA.git ./

	mkdir ./genome

	ln -s ~/GENOMES/Arabidopsis_thaliana/Annotations/Araport-11/Araport11_genes.20151202.gff3 ./genome

	ln -s ~/GENOMES/Arabidopsis_thaliana/Annotations/Araport-11/Araport11_genes.20151202.gtf ./genome

	ln -s ~/GENOMES/Arabidopsis_thaliana/Annotations/Araport-11/At_tair10.fa ./genome

	mkdir fastq-unlev

Place or link the FASTQ files into the above subdirectory. Rename them AtWT_1.fastq, ...etc..., AtWT_17.fastq . Then we need to modify the chromosome labels in the annotation files to match those used in the FASTQ files.

	perl -e 'while(<>){~s/^Chr//g; print}' ./genome/Araport11_genes.20151202.gff3 > tmp.gff3

	perl -e 'while(<>){~s/^C/Pt/g; print}' tmp.gff3 > tmp2.gff3

	perl -e 'while(<>){~s/^M/Mt/g; print}' tmp2.gff3 > ./genome/At_araport11.gff3

	perl -e 'while(<>){~s/^Chr//g; print}' ./genome/Araport11_genes.20151202.gtf > tmp.gtf

	perl -e 'while(<>){~s/^C/Pt/g; print}' tmp.gtf > tmp2.gtf

	perl -e 'while(<>){~s/^M/Mt/g; print}' tmp2.gtf > ./genome/At_araport11.gtf

	rm ./tmp*

## STAR alignment and featureCounts gene expression

Make the STAR index, align the raw FASTQdata, count the reads mapping to genes.

	mkdir ./starindex

	mkdir ./starindex/At_araport11_99bp

	qsub -V -cwd -pe smp 8 -N staridx -b y STAR --runThreadN 8 --runMode genomeGenerate --genomeDir ./starindex/At_araport11_99bp --genomeFastaFiles ./genome/At_tair10.fa --sjdbGTFfile ./genome/At_araport11.gtf --sjdbOverhang 99

	mkdir sam-unlev

	qsub -V -cwd -pe smp 16 -N staral-unlev -t 1-17 ./analysis/star_array.sh -X STAR -c AtWT -i fastq-unlev -a At_araport11.gtf -n At_araport11_99bp -o sam-unlev -t 16

	python3 ./analysis/fileutilities.py T ./sam-unlev/ --dir final | python3 analysis/sequtilities.py P --StarFinalLogs tab -v > ./sam-unlev/AtWT-unlev_summary.tsv

	mkdir ./featurecounts-unlev

	qsub -V cwd -pe smp 4 -N featCnt-unlev -t 1-17 ./analysis/featureCounts_array.sh -X featureCounts -c AtWT -a At_araport11.gtf -i sam-unlev -o featurecounts-unlev -f exon -g gene_id -r 2 -t 4

Collect all the read counts in a single table to use downstream

	mkdir ./combined_counts

	python3 ./analysis/fileutilities.py T ./featurecounts-unlev --dir "tsv$" | perl -e 'while(<>){~s/__Aligned.out.bam.exon\n/\n/;print}' > ./featurecounts-unlev/AtWT-unlev.list

	python3 ./analysis/fileutilities.py L ./featurecounts-unlev/AtWT-unlev.list --cols \-1 -li | perl -e 'while(<>){~s/_\|-1//g;print}' > combined_counts/AtWT-unlev_raw.tsv

## Calculating the correlations between replicates

	mkdir ./results

	Rscript ./analysis/correlations.R AtWT-unlev_raw.tsv > results/AtWT-unlev_raw.tsv_cors.csv

	Rscript ./analysis/levelplot.R results/AtWT-unlev_raw.tsv_cors.tsv

## FDR WT v WT Differential Gene expression (DGE)

This section uses code from the original [Schurch et. al. 2016](https://rnajournal.cshlp.org/content/22/6/839.long) paper to peform WT vs WT DGE calculations for selections of 3-7 replicates in each 'condition', boortstrapped 100 times.First we get the gene identifiers, setup some sub directories and the readcounts for each replicate.

	python3 ./analysis/fileutilities.py T ./combined_counts/AtWT-unlev_raw.tsv -r --cols 0 > At_genes-h.txt

	mkdir DGE_FDR_in

	mkdir ./DGE_FDR_in/AtWTa

	mkdir ./DGE_FDR_in/AtWTb

	mkdir DGE_FDR_out

	python3 ./analysis/fileutilities.py L ./featurecounts-unlev/AtWT-unlev.list -rl --cols 0 6 --out DGE_FDR_in/AtWTa "" '.gbgout'

	python3 ./analysis/fileutilities.py L ./featurecounts-unlev/AtWT-unlev.list -rl --cols 0 6 --out DGE_FDR_in/AtWTb "" '.gbgout'

Then we exclude the "bad" replicate 11.

	rm DGE_FDR_in/AtWTa/AtWT_11.gbgout DGE_FDR_in/AtWTb/AtWT_11.gbgout

Then we perform the DGE bootstrapping...

	bash ./analysis/loop.sh -f 2 -t 7 python ./analysis/grnascripts/Bootstrapping/generic_wrapper.py -o ${PROJECTROOT}/DGE_FDR_out/AtWT_edgeR_100_{val}.sqlite -l ${PROJECTROOT}/DGE_FDR_out/AtWT_edgeR_100_{val}.log -k {val} -r ${PROJECTROOT}/analysis/grnascripts/DE_tool_scripts/edgeR.R --tmpdir=${PROJECTROOT}/NOBACK/tmp-edgeR -n package:default -d ${PROJECTROOT}/DGE_FDR_in -a ${PROJECTROOT}/genome/At_araport11.gff3 -b 100 --precounts --gbgfile ${PROJECTROOT}/analysis/grnascripts/Bootstrapping/group_by_gene.pl --agncfile ${PROJECTROOT}/analysis/grnascripts/Bootstrapping/add_gene_name_column.pl --samtoolspath `which samtools` --Rpath `which Rscript`

	bash ./analysis/loop.sh -f 3 -t 7 python ./analysis/grnascripts/Bootstrapping/generic_wrapper.py -o ${PROJECTROOT}/DGE_FDR_out/AtWT_edgeRglm_100_{val}.sqlite -l ${PROJECTROOT}/DGE_FDR_out/AtWT_edgeRglm_100_{val}.log -k {val} -r ${PROJECTROOT}/analysis/grnascripts/DE_tool_scripts/edgeRglm.R --tmpdir=${PROJECTROOT}/NOBACK/tmp-edgeRglm -n package:default -d ${PROJECTROOT}/DGE_FDR_in -a ${PROJECTROOT}/genome/At_araport11.gff3 -b 100 --precounts --gbgfile ${PROJECTROOT}/analysis/grnascripts/Bootstrapping/group_by_gene.pl --agncfile ${PROJECTROOT}/analysis/grnascripts/Bootstrapping/add_gene_name_column.pl --samtoolspath `which samtools` --Rpath `which Rscript`

	bash ./analysis/loop.sh -f 3 -t 7 python ./analysis/grnascripts/Bootstrapping/generic_wrapper.py -o ${PROJECTROOT}/DGE_FDR_out/AtWT_deseq2_100_{val}.sqlite -l ${PROJECTROOT}/DGE_FDR_out/AtWT_deseq2_100_{val}.log -k {val} -r ${PROJECTROOT}/analysis/grnascripts/DE_tool_scripts/deseq2.R --tmpdir=${PROJECTROOT}/NOBACK/tmp-deseq2 -n package:default -d ${PROJECTROOT}/DGE_FDR_in -a ${PROJECTROOT}/genome/At_araport11.gff3 -b 100 --precounts --gbgfile ${PROJECTROOT}/analysis/grnascripts/Bootstrapping/group_by_gene.pl --agncfile ${PROJECTROOT}/analysis/grnascripts/Bootstrapping/add_gene_name_column.pl --samtoolspath `which samtools` --Rpath `which Rscript`

	bash ./analysis/loop.sh -f 3 -t 7 python ./analysis/grnascripts/Bootstrapping/generic_wrapper.py -o ${PROJECTROOT}/DGE_FDR_out/AtWT_deseq_100_{val}.sqlite -l ${PROJECTROOT}/DGE_FDR_out/AtWT_deseq_100_{val}.log -k {val} -r ${PROJECTROOT}/analysis/grnascripts/DE_tool_scripts/deseq.R --tmpdir=${PROJECTROOT}/NOBACK/tmp-deseq -n package:default -d ${PROJECTROOT}/DGE_FDR_in -a ${PROJECTROOT}/genome/At_araport11.gff3 -b 100 --precounts --gbgfile ${PROJECTROOT}/analysis/grnascripts/Bootstrapping/group_by_gene.pl --agncfile ${PROJECTROOT}/analysis/grnascripts/Bootstrapping/add_gene_name_column.pl --samtoolspath `which samtools` --Rpath `which Rscript`

	bash ./analysis/loop.sh -f 3 -t 7 python ./analysis/grnascripts/Bootstrapping/generic_wrapper.py -o ${PROJECTROOT}/DGE_FDR_out/AtWT_bayseq_100_{val}.sqlite -l ${PROJECTROOT}/DGE_FDR_out/AtWT_bayseq_100_{val}.log -k {val} -r ${PROJECTROOT}/analysis/grnascripts/DE_tool_scripts/bayseq.R --tmpdir=${PROJECTROOT}/NOBACK/tmp-bayseq -n package:default -d ${PROJECTROOT}/DGE_FDR_in -a ${PROJECTROOT}/genome/At_araport11.gff3 -b 100 --precounts --gbgfile ${PROJECTROOT}/analysis/grnascripts/Bootstrapping/group_by_gene.pl --agncfile ${PROJECTROOT}/analysis/grnascripts/Bootstrapping/add_gene_name_column.pl --samtoolspath `which samtools` --Rpath `which Rscript`

	bash ./analysis/loop.sh -f 3 -t 7 python ./analysis/grnascripts/Bootstrapping/generic_wrapper.py -o ${PROJECTROOT}/DGE_FDR_out/AtWT_degseq_100_{val}.sqlite -l ${PROJECTROOT}/DGE_FDR_out/AtWT_degseq_100_{val}.log -k {val} -r ${PROJECTROOT}/analysis/grnascripts/DE_tool_scripts/degseq.R --tmpdir=${PROJECTROOT}/NOBACK/tmp-degseq -n package:default -d ${PROJECTROOT}/DGE_FDR_in -a ${PROJECTROOT}/genome/At_araport11.gff3 -b 100  --precounts --gbgfile ${PROJECTROOT}/analysis/grnascripts/Bootstrapping/group_by_gene.pl --agncfile ${PROJECTROOT}/analysis/grnascripts/Bootstrapping/add_gene_name_column.pl --samtoolspath `which samtools` --Rpath `which Rscript`

	bash ./analysis/loop.sh -f 3 -t 7 python ./analysis/grnascripts/Bootstrapping/generic_wrapper.py -o ${PROJECTROOT}/DGE_FDR_out/AtWT_ebseq_100_{val}.sqlite -l ${PROJECTROOT}/DGE_FDR_out/AtWT_ebseq_100_{val}.log -k {val} -r ${PROJECTROOT}/analysis/grnascripts/DE_tool_scripts/ebseq.R --tmpdir=${PROJECTROOT}/NOBACK/tmp-ebseq -n package:default -d ${PROJECTROOT}/DGE_FDR_in -a ${PROJECTROOT}/genome/At_araport11.gff3 -b 100 --precounts --gbgfile ${PROJECTROOT}/analysis/grnascripts/Bootstrapping/group_by_gene.pl --agncfile ${PROJECTROOT}/analysis/grnascripts/Bootstrapping/add_gene_name_column.pl --samtoolspath `which samtools` --Rpath `which Rscript`

	bash ./analysis/loop.sh -f 3 -t 7 python ./analysis/grnascripts/Bootstrapping/generic_wrapper.py -o ${PROJECTROOT}/DGE_FDR_out/AtWT_limma_100_{val}.sqlite -l ${PROJECTROOT}/DGE_FDR_out/AtWT_limma_100_{val}.log -k {val} -r ${PROJECTROOT}/analysis/grnascripts/DE_tool_scripts/limma.R --tmpdir=${PROJECTROOT}/NOBACK/tmp-limma -n package:default -d ${PROJECTROOT}/DGE_FDR_in -a ${PROJECTROOT}/genome/At_araport11.gff3 -b 100 --precounts --gbgfile ${PROJECTROOT}/analysis/grnascripts/Bootstrapping/group_by_gene.pl --agncfile ${PROJECTROOT}/analysis/grnascripts/Bootstrapping/add_gene_name_column.pl --samtoolspath `which samtools` --Rpath `which Rscript`

	bash ./analysis/loop.sh -f 3 -t 7 python ./analysis/grnascripts/Bootstrapping/generic_wrapper.py -o ${PROJECTROOT}/DGE_FDR_out/AtWT_samseq_100_{val}.sqlite -l ${PROJECTROOT}/DGE_FDR_out/AtWT_samseq_100_{val}.log -k {val} -r ${PROJECTROOT}/analysis/grnascripts/DE_tool_scripts/samseq.R --tmpdir=${PROJECTROOT}/NOBACK/tmp-samseq -n package:default -d ${PROJECTROOT}/DGE_FDR_in -a ${PROJECTROOT}/genome/At_araport11.gff3 -b 100 --precounts --gbgfile ${PROJECTROOT}/analysis/grnascripts/Bootstrapping/group_by_gene.pl --agncfile ${PROJECTROOT}/analysis/grnascripts/Bootstrapping/add_gene_name_column.pl --samtoolspath `which samtools` --Rpath `which Rscript`

	bash ./analysis/loop.sh -f 3 -t 7 python ./analysis/grnascripts/Bootstrapping/generic_wrapper.py -o ${PROJECTROOT}/DGE_FDR_out/AtWT_poissonseq_100_{val}.sqlite -l ${PROJECTROOT}/DGE_FDR_out/AtWT_poissonseq_100_{val}.log -k {val} -r ${PROJECTROOT}/analysis/grnascripts/DE_tool_scripts/poissonseq.R --tmpdir=${PROJECTROOT}/NOBACK/tmp-poissonseq -n package:default -d ${PROJECTROOT}/DGE_FDR_in -a ${PROJECTROOT}/genome/At_araport11.gff3 -b 100 --precounts --gbgfile ${PROJECTROOT}/analysis/grnascripts/Bootstrapping/group_by_gene.pl --agncfile ${PROJECTROOT}/analysis/grnascripts/Bootstrapping/add_gene_name_column.pl --samtoolspath `which samtools` --Rpath `which Rscript`

	bash ./analysis/loop.sh -f 3 -t 7 python ./analysis/grnascripts/Bootstrapping/generic_wrapper.py -o ${PROJECTROOT}/DGE_FDR_out/AtWT_noiseq_100_{val}.sqlite -l ${PROJECTROOT}/DGE_FDR_out/AtWT_noiseq_100_{val}.log -k {val} -r ${PROJECTROOT}/analysis/grnascripts/DE_tool_scripts/noiseq.R --tmpdir=${PROJECTROOT}/NOBACK/tmp-noiseq -n package:default -d ${PROJECTROOT}/DGE_FDR_in -a ${PROJECTROOT}/genome/At_araport11.gff3 -b 100 --precounts --gbgfile ${PROJECTROOT}/analysis/grnascripts/Bootstrapping/group_by_gene.pl --agncfile ${PROJECTROOT}/analysis/grnascripts/Bootstrapping/add_gene_name_column.pl --samtoolspath `which samtools` --Rpath `which Rscript`

With that done we can compute the FDR stats and plot them for the paper...

	mkdir ./DGE_FDR_powerstats

	source ./analyis/loop.sh -v bayseq -v deseq1 -v deseq2 -v degseq -v edger -v edgerglm -v ebseq -v limma -v noiseq -v poissonseq -v samseq "python ./analysis/grnascripts/DE_tool_comparison/make_powerstats_db.pl -test={val} -testinfofile=${PROJECTROOT}/analysis/DGE_FDR_tests_samecond.txt -script=${PROJECTROOT}/analysis/grnascripts/DE_tool_comparison/one_bs_powerstats.pl -genlist=At_genes-h.txt -powerdir=DGE_FDR_powerstats -reffcfile=${PROJECTROOT}/analysis/DGE_FDR_dummy-truth.tsv -maxn=7"

	perl ./analysis/grnascripts/Plotting/compare_null.pl -psfile=results/dge-fdr_bay-deg-de1-de2-eb-edger-eglm-limma-noi-pois-sam.ps -testinfofile=${PROJECTROOT}/analysis/DGE_FDR_tests_samecond.txt -dir=DGE_FDR_powerstats -maxy=1 -tests=bayseq,degseq,deseq1,deseq2,ebseq,edger,edgerglm,limma,noiseq,poissonseq,samseq

## Normalising replicates by sampling reads

Here we downsample the replicates to the lowest replicate so that we can use statistical tests of goodness-of-fit to distributions that that require integer counts. We then Align the data, and get the gene counts (cluding subsetting the replicates for the 'less noisy' set comparison we make in the paper).

	mkdir fastq-lev

	python3 ./analysis/fileutilities.py T ./sam-unlev/AtWT-unlev_summary.tsv --cols 0 2 -rl | perl -e 'while(<>){~s/_/\t/g;print}' > ./combined_counts/AtWT_readcount.dat

	qsub -cwd -V -t 1-17 ./analysis/levelling_array.sh -X analysis/levelling.pl -c AtWT -p 1 -i fastq-unlev -o fastq-lev -n combined_counts/AtWT_readcount.dat

	mkdir sam-lev

	qsub -V -cwd -pe smp 16 -N staral-lev -t 1-17 ./analysis/star_array.sh -X STAR -c AtWT -i fastq-lev -a At_araport11.gtf -n At_araport11_99bp -o sam-lev -t 16

	mkdir featurecounts-lev

	qsub -V cwd -pe smp 4 -N featCnt-lev -t 1-17 ./analysis/featureCounts_array.sh -X featureCounts -c AtWT -a At_araport11.gtf -i sam-lev -o featurecounts-lev -f exon -g gene_id -r 2 -t 4

	python3 ./analysis/fileutilities.py T ./featurecounts-lev --dir "tsv$" | python3 analysis/fileutilities.py P --cols \-1 -lri > ./combined_counts/AtWT-lev_raw.tsv

Collect the counts of the less "noisy" replicates, and for an equally-sized "control" group that includes the "noisy" replicates, to exclude the influence of the number of replicates.

	python3 ./analysis/fileutilities.py T ./combined_counts/AtWT-lev_raw.tsv -ri --cols 1 2 3 4 5 6 7 15 16 17 > ./combined_counts/AtWT-levN_raw.tsv

	python3 ./analysis/fileutilities.py T ./combined_counts/AtWT-lev_raw.tsv -ri --cols 8 9 10 11 12 13 14 15 16 17 > ./combined_counts/AtWT-levC_raw.tsv

## Goodness-of-fit distribution tests

On this data we not run the good-ness of fit tests for the distributions and plot the results for the figures in the paper.

	ln -s /.analysis/defs.dat ./

	qsub -V -cwd -b y perl ./analysis/rnascripts/one_nb_test.pl -batch=1 -batchsize=33851 -outfile=AtWT-lev_nbtest_m_deseq.dat -stat=m -cond=AtWT-lev -norm=deseq -nonzero=1 -type=raw -test=meintanis -ncrit=30 -maxn=10000000

	bash ./analysis/loop.sh -v nb -v norm -v lnorm -v pois 'perl ./analysis/rnascripts/distribution_test.pl -dist={val} -psfile=results/AtWT_{val}_test.ps -cond=AtWT-lev -multicor=bh'

Do the same for the "non-noisy" and "control" subsets as well

	qsub -V -cwd -b y perl ./analysis/rnascripts/one_nb_test.pl -batch=1 -batchsize=33851 -outfile=AtWT-levN_nbtest_m_deseq.dat -stat=m -cond=AtWT-levN -norm=deseq -nonzero=1 -type=raw -test=meintanis -ncrit=30 -maxn=10000000

	bash ./analysis/loop.sh -v nb -v norm -v lnorm -v pois 'perl ./analysis/rnascripts/distribution_test.pl -dist={val} -psfile=results/AtWTn_{val}_test.ps -cond=AtWT-levN -multicor=bh'

	qsub -V -cwd -b y perl ./analysis/rnascripts/one_nb_test.pl -batch=1 -batchsize=33851 -outfile=AtWT-levC_nbtest_m_deseq.dat -stat=m -cond=AtWT-levC -norm=deseq -nonzero=1 -type=raw -test=meintanis -ncrit=30 -maxn=10000000

	bash ./analysis/loop.sh -v nb -v norm -v lnorm -v pois 'perl ./analysis/rnascripts/distribution_test.pl -dist={val} -psfile=results/AtWTc_{val}_test.ps -cond=AtWT-levC -multicor=bh'

And thats it.