Hi,
I want to quantify RNA expression in 10 samples.
So I performed a RNA-seq experiment on Illumina HiSeq 2000 platform (2 x 90 nt).
I used Cole Trapnell method in order to analyse my reads (Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks; Nature Protocols Volume: 7, Pages: 562–578 Year published: (2012) DOI: doi:10.1038/nprot.2012.016 ). The aim of my analysis is to perform a differential analysis focused on well known human transcriptome.
So Cole Trapnell suggest to perform a Tophat alignement followed by a cuffdiff.
I needed to explore each step of this protocol.
Tophat output (accepted_hits.bam) is composed of ~3% of reads mapped at several loci, 85% reads called "properly paired" according to tophat parameters, 2% reads called "not properly paired" with a big insert between both reads and 10% of singletons (reads which lost their mate-pair).
My question is: does cuffdiff include singletons in FPKM calculation?
Thanks by advance,
best
Sophie
I want to quantify RNA expression in 10 samples.
So I performed a RNA-seq experiment on Illumina HiSeq 2000 platform (2 x 90 nt).
I used Cole Trapnell method in order to analyse my reads (Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks; Nature Protocols Volume: 7, Pages: 562–578 Year published: (2012) DOI: doi:10.1038/nprot.2012.016 ). The aim of my analysis is to perform a differential analysis focused on well known human transcriptome.
So Cole Trapnell suggest to perform a Tophat alignement followed by a cuffdiff.
I needed to explore each step of this protocol.
Tophat output (accepted_hits.bam) is composed of ~3% of reads mapped at several loci, 85% reads called "properly paired" according to tophat parameters, 2% reads called "not properly paired" with a big insert between both reads and 10% of singletons (reads which lost their mate-pair).
My question is: does cuffdiff include singletons in FPKM calculation?
Thanks by advance,
best
Sophie
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