Reference Guided Transcript Assembly

######Note on de novo transcript discovery and differential expression using Cufflinks and Cuffdiff ######In the previous module we ran Cufflinks in 'reference only' mode using the '-G/--GTF' Cufflinks option.

######In this module we will run Cufflinks in two additional modes: (1) 'reference guided' mode and (2) 'de novo' mode ######Cufflinks can predict the transcripts present in each library with or without help from knowledge of known transcripts ######Cufflinks will then assign arbitrary transcript IDs to each transcript assembled from the data and estimate expression for those transcripts ######One complication with this method is that in each library a different set of transcripts is likely to be predicted for each library ######There may be a lot of similarities but the number of transcripts and their exact structure will differ in the output files for each library ######Before you can compare across libraries you therefore need to determine which transcripts correspond to each other across the libraries ######Cufflinks provides 'cuffmerge' to combine predicted transcript GTF files from across different libraries ######Once you have a merged GTF file you can run Cuffdiff with this instead of the known transcripts GTF file we used above

######To run Cufflinks in 'reference guided' mode: use the '-g/--GTF-guide' option ######To run Cufflinks in 'de novo' mode do not specify either of the '-G/--GTF' OR '-g/--GTF-guide' options.

######Refer to the Cufflinks manual for a more detailed explanation: ######http://cufflinks.cbcb.umd.edu/manual.html

Cufflinks basic usage:

cufflinks [options] <hits.sam>

######13. CUFFLINKS REFERENCE GUIDED MODE ######Using the alignments we generated in the previous modules we will now run Cuffinks in reference guided mode using the '-g/--GTF-guide' option ######Extra options specified below

'-p 8' tells Cufflinks to use eight CPUs

'-g/--GTF-guide ' Tells Cufflinks to consider known transcript annotations during the assembly process

'-o' tells Cufflinks to write output to a particular directory (one per sample)

cd $RNA_HOME/expression/
mkdir -p tophat_cufflinks/ref_guided
cd tophat_cufflinks/ref_guided

cufflinks -p 8 -o Normal_cDNA1_lib2 --GTF-guide $RNA_HOME/refs/hg19/genes/genes_chr22.gtf --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check $RNA_HOME/alignments/tophat/Normal_cDNA1_lib2/accepted_hits.bam
cufflinks -p 8 -o Normal_cDNA2_lib2 --GTF-guide $RNA_HOME/refs/hg19/genes/genes_chr22.gtf --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check $RNA_HOME/alignments/tophat/Normal_cDNA2_lib2/accepted_hits.bam
cufflinks -p 8 -o Tumor_cDNA1_lib2 --GTF-guide $RNA_HOME/refs/hg19/genes/genes_chr22.gtf --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check $RNA_HOME/alignments/tophat/Tumor_cDNA1_lib2/accepted_hits.bam
cufflinks -p 8 -o Tumor_cDNA2_lib2 --GTF-guide $RNA_HOME/refs/hg19/genes/genes_chr22.gtf --frag-len-mean 262 --frag-len-std-dev 80 --no-update-check $RNA_HOME/alignments/tophat/Tumor_cDNA2_lib2/accepted_hits.bam