Hi,
I performed a detailed time-course mRNA-seq experiment without any sequencing replicates (the input RNA, however, was a pool of equal amounts of total RNA from biological replicates). As I don't have any replicate information for my sequencing data, I refrained from doing any statistical testing.
I used the Tuxedo protocol for data analysis (Trapnel et al, 2012), i.e. read mapping with TopHat2, transcript assembly by Cufflinks for each individual time point, executed Cuffmerge afterwards, and - instead of Cuffdiff, due to the absence of replicates - I ran Cuffquant and Cuffnorm, the later with default settings.
As a result, Cuffnorm outputs sample-normalized FPKM values. It was brought to my attention by Wagner et al (2012), that TPM values are preferable to FPKM values. Converting my FPKM data to TPM values according to https://haroldpimentel.wordpress.com...ression-units/ changes data analysis partly significantly.
I wonder if it is "allowed" to convert cuffnorm-normalized FPKM values to TPM?
And does it make any sense to convert cuffnorm-FPKM values to TPM?
Or is the cuffnorm-normalization already sufficient to enable robust sample-wise comparison of changes in transcript abundances, hence FPKM to TPM conversion would not be necessary?
I performed a detailed time-course mRNA-seq experiment without any sequencing replicates (the input RNA, however, was a pool of equal amounts of total RNA from biological replicates). As I don't have any replicate information for my sequencing data, I refrained from doing any statistical testing.
I used the Tuxedo protocol for data analysis (Trapnel et al, 2012), i.e. read mapping with TopHat2, transcript assembly by Cufflinks for each individual time point, executed Cuffmerge afterwards, and - instead of Cuffdiff, due to the absence of replicates - I ran Cuffquant and Cuffnorm, the later with default settings.
As a result, Cuffnorm outputs sample-normalized FPKM values. It was brought to my attention by Wagner et al (2012), that TPM values are preferable to FPKM values. Converting my FPKM data to TPM values according to https://haroldpimentel.wordpress.com...ression-units/ changes data analysis partly significantly.
I wonder if it is "allowed" to convert cuffnorm-normalized FPKM values to TPM?
And does it make any sense to convert cuffnorm-FPKM values to TPM?
Or is the cuffnorm-normalization already sufficient to enable robust sample-wise comparison of changes in transcript abundances, hence FPKM to TPM conversion would not be necessary?
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