I have 2 genotypes, one resistant and another susceptible with two repeats of each genotype for stress and control condition. I will analyze them with one concentration of heavy metal at two time points in comparison with control genotypes. Briefly two genotypes, two time points, two repeats for heavy metal treatment and control, one heavy metal concentration. Totally 16 samples. could any one give aa advice? Regards
Do not pool replicate samples. This is almost always a mistake. You will need biological replicates for the differential expression analysis later on. You may want to analyze your data with either DESeq2 or EdgeR. Both packages need replicates for proper work.
I am really thankful for your message. You mean that I need at least two replications for differential expression analysis? Morover I repeated plant cultures in three different months. Can I do something with them?
best regards.
Exactly, three or more replicates would even be better. These should be biological sample replicates not simply technical splits of one sample. The reason is that biological variance is much greater than technical.
A good description of the analysis of RNA-Seq data can be found here: http://www.bioconductor.org/help/workflows/rnaseqGene/.
It is also worth to have a look into the papers that describe DESeq2 (Love MI, Huber W and Anders S (2014). “Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.” Genome Biology, 15, pp. 550.) and EdgeR (Robinson MD, McCarthy DJ and Smyth GK (2010). “edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.” Bioinformatics, 26, pp. -1.
McCarthy, J. D, Chen, Yunshun, Smyth and K. G (2012). “Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation.” Nucleic Acids Research, 40(10), pp. -9.).
Regarding the three different cultures: I am not sure whether you can treat them as replicates or not. The growth conditions may differ too much. I cannot give you much advice how to handle them.
I am really grateful for your time and consideration. Your kindly provided suggestions and comments were very useful for me.
Best Regards.
Do not pool unless you barcode the samples. Just keep in mind that if you barcode and pool, you get less RNA reads from each sample. I would say you need at least three reps per genotype per condition.
Thanks a lot for your reply. Would you explain that how could I barcode my samples?
Regards.
best to read this paper here:
http://www.genetics.org/content/185/2/405
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