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  • cuffdiff uses bam file or Sam file

    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.

  • #2
    This is what worries me.

    Code:
    > 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?

    Comment


    • #3
      Whats the code input?

      Comment


      • #4
        Code:
        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

        Comment


        • #5
          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.

          Comment


          • #6
            for more info:
            Code:
            ##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

            Comment

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