cufflinks pipeline

cufflinks.txt

# gather "accepted_hits.bam" to one directory
$ find . -name "accepted_hits.bam" > bam_list
$ while read line; do cp $line  $(dirname $line).bam  ; done < bam_list


# rename bam files to shorten the names (e.g. SAMPLE.trim.cut.fq.gz.bam --> SAMPLE.bam)
$ rename 's/trim.cut.fq.gz.//g' *


# sort/rmdup BAM files (Optional but strongly recommended)
$ for b in ./*.bam; do samtools sort -@ 8 $b $b.sorted ; done
# will produce 'AAA.bam.sorted.bam'


# to remove duplicated reads
$ for b in ./*.sorted.bam; do samtools rmdup -s $b $b.rmdup.bam ; done
# will produce 'AAA.bam.sorted.bam.rmdup.bam'


# cufflinks
# '-G /path/to/genes.gtf' let cufflinks count reads mapped only 
# on known (reference) transcripts.
$ mkdir cufflinks
$ for b in BAM/*.rmdup.bam; do i=$(basename $b); echo ' >>> ' $i ; echo;\
cufflinks -p 8  --library-type fr-firststrand -G ~/00-NGS/Annotation/Bos_taurus_UCSC_bosTau8/GTF/genes.gtf \
-o cufflinks/$i $b; echo;\
done


# generate 'assembly.txt' file
$ find . -name "transcripts.gtf" > assembly.txt
$ cat assembly.txt
/path/to/Sample1/transcripts.gtf
/path/to/Sample2/transcripts.gtf
/path/to/Sample3/transcripts.gtf
/path/to/Sample4/transcripts.gtf


# cuffmerge
$ cuffmerge -p 8 -g genes.gtf assembly.txt

# you can go directly to 'cuffdiff' or do 'cuffquant --> cuffdiff or cuffnorm'

# cuffquant
$ for b in ../BAM/*.rmdup.bam; do i=$(basename $b); echo ' >>> ' $i ; echo;\
cufflinks -p 8 merged_asm/merged.gtf -o cufflinks/$i $b; echo;  done

# generate sample_sheet
$ find . -name "abundances.cxb" > sample_sheet
# or
$ for b in /PATH/TO/*.bam;
do
echo $b >> sample_seet
done

# sample_sheet should look like below
# the code above generates only 'sample_id'
# add group names manually
   sample group
   /path/to/C1_R1.sam C1
   /path/to/C1_R2.sam C1
   /path/to/C2_R1.sam C2
   /path/to/C2_R2.sam C2
 or 
   sample group
   /path/to/C1_R1.cxb C1
   /path/to/C1_R2.cxb C1
   /path/to/C2_R1.cxb C2
   /path/to/C2_R2.cxb C2

# cuffnorm
$ cuffnorm -p 8  --library-type fr-firststrand --use-sample-sheet ../merged_asm/merged.gtf -o norm_out sample_sheet

# cuffdiff
$ cuffdiff -p 8  --library-type fr-firststrand --use-sample-sheet ../merged_asm/merged.gtf -o diff_out/ sample_sheet