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Obi Griffith edited this page Nov 13, 2016 · 1 revision

RNA-seq Flowchart - Module 4

#3-ii. Differential Expression Use Cuffmerge and Cuffdiff to compare the tumor and normal conditions. Refer to the Cufflinks manual for a more detailed explanation:

Cuffmerge basic usage:

cuffmerge [options]* <assembly_GTF_list.txt>
  • "assembly_GTF_list.txt" is a text file "manifest" with a list (one per line) of GTF files that you would like to merge together into a single GTF file.

Extra options specified below:

  • '-p 8' tells cuffmerge to use eight CPUs
  • '-o' tells cuffmerge to write output to a particular directory
  • '-g' tells cuffmerge where to find reference gene annotations. It will use these annotations to gracefully merge novel isoforms (for de novo runs) and known isoforms and maximize overall assembly quality.
  • '-s' tells cuffmerge where to find the reference genome files

Merge all 6 cufflinks results so that they will have the same set of transcripts for comparison purposes

cd $RNA_HOME/expression/tophat_cufflinks/ref_only/
ls -1 *Rep*ERCC*/transcripts.gtf > assembly_GTF_list.txt
cuffmerge -p 8 -o merged -g $RNA_HOME/refs/hg19/genes/genes_chr22_ERCC92.gtf -s $RNA_HOME/refs/hg19/bwt/chr22_ERCC92/ assembly_GTF_list.txt

###OPTIONAL ALTERNATIVE - Merge Cufflinks GTFs for STAR Perform the merge step for STAR-alignment-based cufflinks output:

cd $RNA_HOME/expression/star_cufflinks/ref_only/
ls -1 *Rep*ERCC*/transcripts.gtf > assembly_GTF_list.txt
cuffmerge -p 8 -o merged -g $RNA_HOME/refs/hg19/genes/genes_chr22_ERCC92.gtf -s $RNA_HOME/refs/hg19/fasta/chr22_ERCC92/ assembly_GTF_list.txt

####END OF OPTIONAL ALTERNATIVE - Merge Cufflinks GTFs for STAR

###OPTIONAL ALTERNATIVE - Merge Cufflinks GTFs for HISAT2 Perform the merge step for HISAT2-alignment-based cufflinks output:

cd $RNA_HOME/expression/hisat2_cufflinks/ref_only/
ls -1 *Rep*ERCC*/transcripts.gtf > assembly_GTF_list.txt
cuffmerge -p 8 -o merged -g $RNA_HOME/refs/hg19/genes/genes_chr22_ERCC92.gtf -s $RNA_HOME/refs/hg19/fasta/chr22_ERCC92/ assembly_GTF_list.txt

####END OF OPTIONAL ALTERNATIVE - Merge Cufflinks GTFs for HISAT2

Cuffdiff basic usage:

cuffdiff [options] <transcripts.gtf> <sample1_hits.sam> <sample2_hits.sam> [... sampleN_hits.sam]
  • Supply replicate SAMs as comma separated lists for each condition:
  • Example: sample1_rep1.sam,sample1_rep2.sam,...sample1_repM.sam
  • '-p 8' tells cuffdiff to use eight CPUs
  • '-L' tells cuffdiff the labels to use for samples

Create necessary directories:

cd $RNA_HOME/
mkdir -p de/tophat_cufflinks/ref_only
cd $RNA_HOME/alignments/tophat/

Generate the cuffquant binary format files for cuffdiff

cuffquant -p 8 --library-type fr-firststrand --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check -o UHR_Rep1_ERCC-Mix1 $RNA_HOME/expression/tophat_cufflinks/ref_only/merged/merged.gtf UHR_Rep1_ERCC-Mix1/accepted_hits.bam
cuffquant -p 8 --library-type fr-firststrand --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check -o UHR_Rep2_ERCC-Mix1 $RNA_HOME/expression/tophat_cufflinks/ref_only/merged/merged.gtf UHR_Rep2_ERCC-Mix1/accepted_hits.bam
cuffquant -p 8 --library-type fr-firststrand --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check -o UHR_Rep3_ERCC-Mix1 $RNA_HOME/expression/tophat_cufflinks/ref_only/merged/merged.gtf UHR_Rep3_ERCC-Mix1/accepted_hits.bam

cuffquant -p 8 --library-type fr-firststrand --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check -o HBR_Rep1_ERCC-Mix2 $RNA_HOME/expression/tophat_cufflinks/ref_only/merged/merged.gtf HBR_Rep1_ERCC-Mix2/accepted_hits.bam
cuffquant -p 8 --library-type fr-firststrand --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check -o HBR_Rep2_ERCC-Mix2 $RNA_HOME/expression/tophat_cufflinks/ref_only/merged/merged.gtf HBR_Rep2_ERCC-Mix2/accepted_hits.bam
cuffquant -p 8 --library-type fr-firststrand --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check -o HBR_Rep3_ERCC-Mix2 $RNA_HOME/expression/tophat_cufflinks/ref_only/merged/merged.gtf HBR_Rep3_ERCC-Mix2/accepted_hits.bam

Perform UHR vs. HBR comparison, using all replicates, for known (reference only mode) transcripts:

cuffdiff -p 8 -L UHR,HBR -o $RNA_HOME/de/tophat_cufflinks/ref_only/ --library-type fr-firststrand --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check $RNA_HOME/expression/tophat_cufflinks/ref_only/merged/merged.gtf UHR_Rep1_ERCC-Mix1/abundances.cxb,UHR_Rep2_ERCC-Mix1/abundances.cxb,UHR_Rep3_ERCC-Mix1/abundances.cxb HBR_Rep1_ERCC-Mix2/abundances.cxb,HBR_Rep2_ERCC-Mix2/abundances.cxb,HBR_Rep3_ERCC-Mix2/abundances.cxb

###OPTIONAL ALTERNATIVE - CuffDiff for STAR results perform the cuffdiff step for STAR-alignment-based cuffmerge output:

cd $RNA_HOME/
mkdir -p de/star_cufflinks/ref_only
cd $RNA_HOME/alignments/star/

cuffquant -p 8 --library-type fr-firststrand --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check -o UHR_Rep1 $RNA_HOME/expression/star_cufflinks/ref_only/merged/merged.gtf UHR_Rep1/Aligned.out.sorted.bam
cuffquant -p 8 --library-type fr-firststrand --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check -o UHR_Rep2 $RNA_HOME/expression/star_cufflinks/ref_only/merged/merged.gtf UHR_Rep2/Aligned.out.sorted.bam
cuffquant -p 8 --library-type fr-firststrand --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check -o UHR_Rep3 $RNA_HOME/expression/star_cufflinks/ref_only/merged/merged.gtf UHR_Rep3/Aligned.out.sorted.bam

cuffquant -p 8 --library-type fr-firststrand --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check -o HBR_Rep1 $RNA_HOME/expression/star_cufflinks/ref_only/merged/merged.gtf HBR_Rep1/Aligned.out.sorted.bam
cuffquant -p 8 --library-type fr-firststrand --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check -o HBR_Rep2 $RNA_HOME/expression/star_cufflinks/ref_only/merged/merged.gtf HBR_Rep2/Aligned.out.sorted.bam
cuffquant -p 8 --library-type fr-firststrand --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check -o HBR_Rep3 $RNA_HOME/expression/star_cufflinks/ref_only/merged/merged.gtf HBR_Rep3/Aligned.out.sorted.bam

cuffdiff -p 8 -L UHR,HBR -o $RNA_HOME/de/star_cufflinks/ref_only/ --library-type fr-firststrand --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check $RNA_HOME/expression/star_cufflinks/ref_only/merged/merged.gtf UHR_Rep1/abundances.cxb,UHR_Rep2/abundances.cxb,UHR_Rep3/abundances.cxb HBR_Rep1/abundances.cxb,HBR_Rep2/abundances.cxb,HBR_Rep3/abundances.cxb

####END OF OPTIONAL ALTERNATIVE - CuffDiff for STAR results

###OPTIONAL ALTERNATIVE - CuffDiff for HISAT2 results perform the cuffdiff step for HISAT2-alignment-based cuffmerge output:

cd $RNA_HOME/
mkdir -p de/hisat2_cufflinks/ref_only
cd $RNA_HOME/alignments/hisat2/

cuffquant -p 8 --library-type fr-firststrand --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check -o UHR_Rep1 $RNA_HOME/expression/hisat2_cufflinks/ref_only/merged/merged.gtf UHR_Rep1/Aligned.out.sorted.bam
cuffquant -p 8 --library-type fr-firststrand --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check -o UHR_Rep2 $RNA_HOME/expression/hisat2_cufflinks/ref_only/merged/merged.gtf UHR_Rep2/Aligned.out.sorted.bam
cuffquant -p 8 --library-type fr-firststrand --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check -o UHR_Rep3 $RNA_HOME/expression/hisat2_cufflinks/ref_only/merged/merged.gtf UHR_Rep3/Aligned.out.sorted.bam

cuffquant -p 8 --library-type fr-firststrand --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check -o HBR_Rep1 $RNA_HOME/expression/hisat2_cufflinks/ref_only/merged/merged.gtf HBR_Rep1/Aligned.out.sorted.bam
cuffquant -p 8 --library-type fr-firststrand --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check -o HBR_Rep2 $RNA_HOME/expression/hisat2_cufflinks/ref_only/merged/merged.gtf HBR_Rep2/Aligned.out.sorted.bam
cuffquant -p 8 --library-type fr-firststrand --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check -o HBR_Rep3 $RNA_HOME/expression/hisat2_cufflinks/ref_only/merged/merged.gtf HBR_Rep3/Aligned.out.sorted.bam

cuffdiff -p 8 -L UHR,HBR -o $RNA_HOME/de/hisat2_cufflinks/ref_only/ --library-type fr-firststrand --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check $RNA_HOME/expression/hisat2_cufflinks/ref_only/merged/merged.gtf UHR_Rep1/abundances.cxb,UHR_Rep2/abundances.cxb,UHR_Rep3/abundances.cxb HBR_Rep1/abundances.cxb,HBR_Rep2/abundances.cxb,HBR_Rep3/abundances.cxb

####END OF OPTIONAL ALTERNATIVE - CuffDiff for HISAT2 results

What does the raw output from Cuffdiff look like?

cd $RNA_HOME/de/tophat_cufflinks/ref_only
ls -l
head isoform_exp.diff
grep -P "gene_id|OK" isoform_exp.diff | cut -f 2-6,8-10,12 | sort -k 9,9 | less -S

Press 'q' to exit the 'less' display

How many genes are there on this chromosome?

grep -v gene_id gene_exp.diff | wc -l

How many were detected above 0 in UHR or HBR (take the sum of expression values for both and check for greater than 0)?

grep -v gene_id gene_exp.diff | perl -ne '@line=split("\t", $_); $sum=$line[7]+$line[8]; if ($sum > 0){print "$sum\n";}' | wc -l

How many differentially expressed genes were found on this chromosome (p-value < 0.05)?

grep -v gene_id gene_exp.diff | cut -f 12 | perl -ne 'if ($_ < 0.05){print "$_"}' | wc -l

Display the top 20 DE genes. Look at some of those genes in IGV - do they make sense?

grep -P "OK|gene_id" gene_exp.diff | sort -k 12n,12n | head -n 20 | cut -f 3,5,6,8,9,10,12,13,14

Save all genes with P<0.05 to a new file.

grep -P "OK|gene_id" gene_exp.diff | sort -k 12n,12n | cut -f 3,5,6,8,9,10,12,13,14 | perl -ne '@data=split("\t", $_); if ($data[6]<=0.05){print;}' > DE_genes.txt

| Previous Section | This Section | Next Section | |:-------------------------------:|:---------------------------------------------------:|:-----------------------------------------:| | Expression | Differential Expression | DE Visualization |

##Note: The current version of this tutorial is now at www.rnaseq.wiki

Table of Contents
Module 0: Authors | Citation | Syntax | Intro to AWS | Log into AWS | Unix | Environment | Resources
Module 1: Installation | Reference Genomes | Annotations | Indexing | Data | Data QC
Module 2: Adapter Trim | Alignment | IGV | Alignment Visualization | Alignment QC
Module 3: Expression | Differential Expression | DE Visualization
Module 4: Ref Guided | De novo | Merging | Differential Splicing | Splicing Visualization
Module 5: Kallisto
Appendix: Abbreviations | Lectures | Practical Exercise Solutions | Integrated Assignment | Proposed Improvements | AWS Setup

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