Hi there,
I'm finally posting rather than viewing as i'd appreciate some advice about my big experiment.
I am looking at the interaction of hexaploid wheat and a fungal pathogen Claviceps purpurea and have completed an RNA-seq experiment comparing inoculated vs mock inoculated with three tissue types, five timepoints and I have 2-3 biological reps for each of these. The libraries were SE 100bp Illumina sequenced and have kindly been analysed by our collaborative partner whereby he mapped them sequentially to a wheat UniGene reference set then to a Claviceps predicted coding region reference set.
We have a table of mapped read numbers and the resulting RPKM values for each of the libraries, but we have also performed 2-way ANOVA so that we have mean RPKM values for each time-point and tissue type which we’ve sorted based on interaction P-value for inoculated vs mock. I would also like to use tools such as EdgeR or DEGseq to check that the same genes are differentially expressed.
So, my questions are:
1. It is possible to import all of the raw read numbers into EdgeR or DEGseq and then analyse in a pairwise way? Should I compare a single mock inoculated (say at Tzero) with all of the other inoculated libraries for that tissue? Is there scope for analysing more of the experiment in one go to get an idea of how a gene performs in a single tissue type over time?
2. I would love to be able to perform a cluster analysis and see the lists of genes that fit into each cluster. I’d also like to easily view a MyFavouriteGeneList (we probably all have one right??) to see what cluster they fall into so that I can possibly use this to identify lists of co-regulated genes. Any ideas on how to do this –on the RPKMs I already have or the outputs from EdgeR?
3. I would then like to import a transformed data set into something like Cytoscape so that I can then play around with visualising networks of DE genes and include (patchy) GO annotation for the wheat reference. What plug-ins would you recommend?
I think I’ll leave my wish-list there for now. Any suggestions gratefully received.. I should also mention i've used R for QTL work but never sequence analysis so i'm a bit of a newbie
Thanks,
Anna
I'm finally posting rather than viewing as i'd appreciate some advice about my big experiment.
I am looking at the interaction of hexaploid wheat and a fungal pathogen Claviceps purpurea and have completed an RNA-seq experiment comparing inoculated vs mock inoculated with three tissue types, five timepoints and I have 2-3 biological reps for each of these. The libraries were SE 100bp Illumina sequenced and have kindly been analysed by our collaborative partner whereby he mapped them sequentially to a wheat UniGene reference set then to a Claviceps predicted coding region reference set.
We have a table of mapped read numbers and the resulting RPKM values for each of the libraries, but we have also performed 2-way ANOVA so that we have mean RPKM values for each time-point and tissue type which we’ve sorted based on interaction P-value for inoculated vs mock. I would also like to use tools such as EdgeR or DEGseq to check that the same genes are differentially expressed.
So, my questions are:
1. It is possible to import all of the raw read numbers into EdgeR or DEGseq and then analyse in a pairwise way? Should I compare a single mock inoculated (say at Tzero) with all of the other inoculated libraries for that tissue? Is there scope for analysing more of the experiment in one go to get an idea of how a gene performs in a single tissue type over time?
2. I would love to be able to perform a cluster analysis and see the lists of genes that fit into each cluster. I’d also like to easily view a MyFavouriteGeneList (we probably all have one right??) to see what cluster they fall into so that I can possibly use this to identify lists of co-regulated genes. Any ideas on how to do this –on the RPKMs I already have or the outputs from EdgeR?
3. I would then like to import a transformed data set into something like Cytoscape so that I can then play around with visualising networks of DE genes and include (patchy) GO annotation for the wheat reference. What plug-ins would you recommend?
I think I’ll leave my wish-list there for now. Any suggestions gratefully received.. I should also mention i've used R for QTL work but never sequence analysis so i'm a bit of a newbie
Thanks,
Anna