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Bash script to calculate FPKMs from the Human BodyMap 2.0 mRNA-seq data.
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| # Eric Minikel | |
| # CureFFI.org | |
| # 2013-07-04 | |
| # Bash script to generate FPKMs from Ensembl Human Bodymap 2.0 RNA-seq data | |
| # Announcement of Human BodyMap 2.0 data availability: http://www.ensembl.info/blog/2011/05/24/human-bodymap-2-0-data-from-illumina/ | |
| # BAM list: ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/ | |
| # STEP 1: DOWNLOAD THE BAMS FROM ENSEMBL | |
| mkdir /data/HD/dataset/humanbodymap2.0 | |
| cat > wget.bash | |
| #!/bin/bash | |
| cd /data/HD/dataset/humanbodymap2.0 | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.adipose.1.bam | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.adipose.1.bam.bai | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.adrenal.1.bam | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.adrenal.1.bam.bai | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.blood.1.bam | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.blood.1.bam.bai | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.brain.1.bam | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.brain.1.bam.bai | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.breast.1.bam | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.breast.1.bam.bai | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.colon.1.bam | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.colon.1.bam.bai | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.heart.1.bam | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.heart.1.bam.bai | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.kidney.1.bam | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.kidney.1.bam.bai | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.liver.1.bam | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.liver.1.bam.bai | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.lung.1.bam | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.lung.1.bam.bai | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.lymph.1.bam | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.lymph.1.bam.bai | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.merged.1.bam | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.merged.1.bam.bai | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.ovary.1.bam | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.ovary.1.bam.bai | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.pooled.1.bam | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.pooled.1.bam.bai | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.prostate.1.bam | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.prostate.1.bam.bai | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.skeletal_muscle.1.bam | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.skeletal_muscle.1.bam.bai | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.testes.1.bam | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.testes.1.bam.bai | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.thyroid.1.bam | |
| wget ftp://ftp.ensembl.org/pub/release-70/bam/homo_sapiens/genebuild/GRCh37.HumanBodyMap.thyroid.1.bam.bai | |
| bsub -q long -W 160:00 < wget.bash | |
| cd /data/HD/dataset/gtf | |
| wget ftp://ftp.ensembl.org/pub/release-70/gtf/homo_sapiens/Homo_sapiens.GRCh37.70.gtf.gz | |
| gunzip Homo_sapiens.GRCh37.70.gtf.gz | |
| # STEP 2: REMOVE @RG TAGS TO MAKE BAMS COMPATIBLE WITH CUFFLINKS** (see below) | |
| cd /data/HD/dataset/humanbodymap2.0 | |
| mkdir no-rg | |
| for file in GRCh37.*.bam | |
| do | |
| bsub -q medium "cd /data/HD/dataset/humanbodymap2.0; samtools view -h $file | grep -v ^@RG | samtools view -Sbh - > no-rg/$file" | |
| done | |
| bsub -q medium "cd /data/HD/dataset/humanbodymap2.0; samtools view -h $file | grep -v ^@RG | samtools view -Sbh - > no-rg/$file" | |
| # STEP 3: QUANTIFY KNOWN (REFSEQ) TRANSCRIPTS WITH CUFFLINKS | |
| cd /data/HD/dataset/humanbodymap2.0 | |
| for file in *.bam | |
| do | |
| bsub -q long -W 160:00 "cd /data/HD/dataset/humanbodymap2.0; cufflinks -o ./cufflinks/$file -G /data/HD/dataset/gtf/Homo_sapiens.GRCh37.70.gtf ./no-rg/$file" | |
| done | |
| # STEP 4: GROUP CUFFLINKS OUTPUT ACROSS TISSUES | |
| cd /data/HD/dataset/humanbodymap2.0 | |
| # prep header rows | |
| cat cufflinks/GRCh37.HumanBodyMap.adipose.1.bam/isoforms.fpkm_tracking | head -1 | awk '{print "TISSUE\t"$0}' > combined/all.isoforms.fpkm_tracking | |
| cat cufflinks/GRCh37.HumanBodyMap.adipose.1.bam/genes.fpkm_tracking | head -1 | awk '{print "TISSUE\t"$0}' > combined/all.genes.fpkm_tracking | |
| # combine files | |
| for tissue in {adipose,adrenal,blood,brain,breast,colon,heart,kidney,liver,lung,lymph,ovary,prostate,skeletal_muscle,testes,thyroid} | |
| do | |
| # combine isoforms data into one file, adding a column for tissue type | |
| cat cufflinks/GRCh37.HumanBodyMap.$tissue.1.bam/isoforms.fpkm_tracking | tail -n +2 | awk -v tissue=$tissue '{print tissue"\t"$0}' >> combined/all.isoforms.fpkm_tracking | |
| # combine gene symbol data into one file, adding a column for tissue type | |
| cat cufflinks/GRCh37.HumanBodyMap.$tissue.1.bam/genes.fpkm_tracking | tail -n +2 | awk -v tissue=$tissue '{print tissue"\t"$0}' >> combined/all.genes.fpkm_tracking | |
| done | |
| # STEP 5: CAST INTO MATRIX | |
| R # open R and run rest of code in R | |
| require(reshape2) | |
| genes = read.table('combined/all.genes.fpkm_tracking',header=TRUE) | |
| gene.matrix = acast(data = genes, formula = gene_short_name ~ TISSUE, fun.aggregate=sum, value.var="FPKM") | |
| write.csv(gene.matrix,'combined/gene.matrix.csv',row.names=TRUE,quote=TRUE) | |
| isoforms = read.table('combined/all.isoforms.fpkm_tracking',header=TRUE) | |
| isoform.matrix = acast(data = isoforms, formula = tracking_id ~ TISSUE, fun.aggregate=mean, value.var="FPKM") # fun.aggregate doesn't matter b/c no duplicates | |
| write.csv(isoform.matrix,'combined/isoform.matrix.csv',row.names=TRUE,quote=TRUE) | |
| ##### ** re: Step 2 - some quick notes about the debugging process for figuring out why cufflinks wouldn't work with the Human BodyMap BAMs: | |
| # # creating a smaller file to use for debugging | |
| # samtools view -h GRCh37.HumanBodyMap.prostate.1.bam GL000210.1 | samtools view -Sbh - -o prostate.gl.bam | |
| # | |
| # # debugging: figuring out that the presence of the single line is sufficient to reproduce segmentation fault | |
| # # offending line: @RG ID:prostate_75_fcb PL: PU: ST: LB: DS: SM:prostate CN: | |
| # samtools view -h prostate.gl.bam | grep -v ID:prostate_75_fcb > prostate.gl.1rg.sam | |
| # samtools view -Sbh prostate.gl.1rg.sam > prostate.gl.1rg.bam | |
| # cufflinks prostate.gl.1rg.bam # actually works | |
| # | |
| # samtools view -h prostate.gl.bam | grep -v ID:prostate_50_fca_2 > prostate.gl.otherrg.sam | |
| # samtools view -Sbh prostate.gl.otherrg.sam > prostate.gl.otherrg.bam | |
| # cufflinks prostate.gl.otherrg.bam # totally does not work |
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