cuffdiff uses bam file or Sam file

[wilson90]

Just wondering if cuffdiff uses Sam file or bam file as input.

The reason for my post is because in my previous run, the output of cuffdiff says no differential expression is performed on my data. So I wonder if it is because I am providing bam instead of Sam as input.

[wilson90]

This is what worries me.

> Processed 28454 loci.                        [*************************] 100%
Performed 0 isoform-level transcription difference tests
Performed 0 tss-level transcription difference tests
Performed 0 gene-level transcription difference tests
Performed 0 CDS-level transcription difference tests
Performed 0 splicing tests
Performed 0 promoter preference tests
Performing 0 relative CDS output tests
Writing isoform-level FPKM tracking
Writing TSS group-level FPKM tracking
Writing gene-level FPKM tracking
Writing CDS-level FPKM tracking
Writing isoform-level count tracking
Writing TSS group-level count tracking
Writing gene-level count tracking
Writing CDS-level count tracking
Writing isoform-level read group tracking
Writing TSS group-level read group tracking
Writing gene-level read group tracking
Writing CDS-level read group tracking
Writing read group info
Writing run info

Why no differential test is being performed?

[sindrle]

Whats the code input?

[wilson90]

cuffdiff -o bam_noslash -b $GENOME -p 35 -L G1,G2 -N -u merged_asm/merged.gtf $BAM1,$BAM2,$BAM3,$BAM4,$BAM5,$BAM6,$BAM7,$BAM8,$BAM9,$BAM10 $BAM11,$BAM12,$BAM13,$BAM14,$BAM15,$BAM16,$BAM17,$BAM18,$BAM19
,$BAM20

[wilson90]

Would it be because:
1. Annotation issue?
2. I remove duplicative reads using picard?
3. Library issue ( among biological replicates)?
4. I have performed upper-quartile normalization in Cufflinks, does it affect?

Thank you.

[wilson90]

for more info:

##Trimming Low Qual reads and Bases with low quality
java -jar Trimmomatic-0.30/trimmomatic-0.30.jar PE -phred33 read_1.fq read_2.fq output_forward_paired.fq.gz output_forward_unpaired.fq.gz output_reverse_paired.fq.gz output_reve
rse_unpaired.fq.gz LEADING:28 TRAILING:28 SLIDINGWINDOW:4:15 MINLEN:50



##Checking Quality
gunzip output_forward_paired.fq.gz
gunzip output_reverse_paired.fq.gz
fastqc --extract -f fastq output_forward_paired.fq output_reverse_paired.fq


##Running Tophat for mapping
tophat2 -p 35 bowtie2/mm9 output_forward_paired.fq output_reverse_paired.fq 


##Running removal of PCR duplicates.
cd tophat_out
PICARD=/picard-tools-1.91/MarkDuplicates.jar
BAM=accepted_hits.bam
java -jar $PICARD INPUT=$BAM OUTPUT=nodup.bam METRICS_FILE=dup.txt VALIDATION_STRINGENCY=LENIENT REMOVE_DUPLICATES=true TMP_DIR=/tmp


##Running cufflinks to quantify transcript without transcript discovery
mkdir cuff_no_novel
cd cuff_no_novel
BAM=../nodup.bam
cufflinks --num-threads 35 --quiet --no-update-check --frag-bias-correct --multi-read-correct  --upper-quartile-norm -G gtf/mm9_annotation.gtf -b bowtie2_path/base/mm9.fa $BAM