Analysing Transcriptome with CLC - how to do it the best way?
Hi,
I hope someone can help me ... I'm really new to mRNAseq, statistical anaylsis and so on (and I don't really understand what I'm doing...
)
I have to analyse my data and I use CLC. For my experiment I have prepared 36 libraries. I harvested two different tissues (leaves and roots of maize) of 3 lines at 3 timepoints in two experiments. So far I worked only with the leaf samples.
I have set up an experiment where I mapped the reads against the transcripts. Now I have a problem with the analysis. I want to detect differential expressed genes and I need to filter for background transcription. As I have already an interesting gene which is low expressed, my cutoff would be 1 RPKM. 1. Question: Does it makes sense to filter for the means of the RPKMS from the replicates or is it better to filter for each library?
I would like to use the DGE package of CLC to find the differential expressed genes. Now comes my 2. question: Do I have to run the test before filtering or after filtering for the RPKM? I think somehow the test includes the library size and if I filter for the RPKM, not all of the library is included...
Thank you very much for your answers!!!
Kind regards
Dobbit
I hope someone can help me ... I'm really new to mRNAseq, statistical anaylsis and so on (and I don't really understand what I'm doing...
)I have to analyse my data and I use CLC. For my experiment I have prepared 36 libraries. I harvested two different tissues (leaves and roots of maize) of 3 lines at 3 timepoints in two experiments. So far I worked only with the leaf samples.
I have set up an experiment where I mapped the reads against the transcripts. Now I have a problem with the analysis. I want to detect differential expressed genes and I need to filter for background transcription. As I have already an interesting gene which is low expressed, my cutoff would be 1 RPKM. 1. Question: Does it makes sense to filter for the means of the RPKMS from the replicates or is it better to filter for each library?
I would like to use the DGE package of CLC to find the differential expressed genes. Now comes my 2. question: Do I have to run the test before filtering or after filtering for the RPKM? I think somehow the test includes the library size and if I filter for the RPKM, not all of the library is included...
Thank you very much for your answers!!!
Kind regards
Dobbit