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  • several questions about tophat and cufflinks

    1. for paired-end rna seq reads, how does --mate-inner-dist and --mate-std-dev affect the tophat mapping result? According to my understanding, these two parameters should only affect the second column of sam/bam output.

    2. for cufflinks package, why cufflinks and cuffdiff give inconsistent fpkm value for some transcripts? Below is my command to run cufflinks and cuffdiff
    Cufflinks -G ensembl_hg19.gtf t1_tophat_hits.bam
    cuffdiff -p 20 --total-hits-norm ensembl_hg19.gtf r1_tophat_hits.bam r2_tophat_hits.bam

    3. if a particular loci has several isoforms, for example iso1,iso2,..., iso5, but the sample sequenced only contains two of the five isoforms, such as iso1 and iso2. If I use gtf file conteining all 5 isoforms annotation to run cufflinks, whether the nonexistent three isoforms will affect the expression abundance estimation for actually existent isoforms (iso1 and iso2)? Can cufflinks give 0 fpkm for these 3 nonexistent isoforms?

    Thanks in advance and look forward to your response.

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