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8.1 years ago
biomoon
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10
How to calculate RPKM using cuffdiff after obtaining accepted_hits.bam? Any suggestion will be appreciated. Thanks very much!
Thank you for your suggestion. Can you show me the command line of Cuffdiff with accepted_hits.bam and a genome reference GTF? Thanks a lot!
Read the manual, it's all there. The flow chart shows the typical route from alignments to differential expression.
Regardless of that flow chart, is that possible that I only use genome reference GTF and bam file to get FPKM using only Cuffdiff? Show me if you know. Thanks.
Regardless of that flowchart - This is the manual for cuffdiff, which performs the statistical tests to calculate FPKM and perform differential expression from your alignments and gtf. All the command line arguments and options are there.
I have seen that manual before I posted the disscussion. I am just curious how the author of one paper empoy Cufflink to calculate RPKM (or FPKM) in one software. The input of that software is alignment results of RNA-seq and a reference genome annotation. They may changed the algorithm. I think some people may know. Anyway, thank you for your reply! I appreciate it.
Cufflinks will calculate FPKM on a per sample basis, it's a transcriptome assembler, and a key element to that will be the quantification of how much stuff is assembled. I don't think the methodology has changed drastically, if at all - Cufflink's last update was its move to Github in 2014. Cufflinks outputs transcript and gene level FPKM tracking file, along with the GTF file.