Hi All,
I hope you are all safe and healthy. Basically, I have no experience with RNAseq data analysis and I have received some data in the ".genes.fpkm_tracking" and ".isoforms.fpkm_tracking" format and have no idea how to extract them to create a heatmap for DEGs. I really appreciate it if someone could provide some instruction or tell me where to look to find such instruction.
Take Care, Hamid
1 answer
These files come out of CuffDiff. To extract the differentially expressed genes, CuffDiff should also have output a file called gene_exp.diff. If you need to call differential expression yourself, CuffDiff also outputs files call gene.count_tracking which can be used with DEseq or edgeR to call differential expression.
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Welcome to the crazy world of RNAseq. You should really try to get your hands on raw counts. FPKM is not comparable between samples and most robust DE analysis is performed on raw counts.
Heatmaps may come into play much later. DE analysis gives you statistically significant DE genes while heatmaps are a fancy way of showing nothing in particular.
The main problem here is that these "formats" are just file suffixes that someone assigned to them, this is no official format, and therefore there is probably no documentation on it. Unless the script to generate these tables is available and reviewable to you I'd strongly recommend to trash it. It would be irreproducible without the code. Better get your hands on the raw counts and follow a vignette such as DESeq2 for DE testing, or, if only like fpkm is available, use the limma-trend pipeline.
Actaully, I do know what these are - these are the output files from CuffDiff.
Hi, while I agree with the two comments above (be careful with that data if you don't know how it was generated), have you tried, out of curiosity, to open the files with a simple text editor (notepad, textedit, etc...) ? More often than not, fancy file extension in bioinformatics are just text file.