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.
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.