Hi all,
I'm relatively new to RNA-seq analysis and want to perform differential expression analysis (DEA) of lncRNAs. I have read multiple different protocols and I think the following method would be valid and I hoped to receive feedback on it:
- calculate RPKM values from the raw counts from htseq-count
- remove genes with 50th-percentile RPKM =0, and then only keep the genes with 90th-percentile RPKM < 0.1 as suggested here (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4777353/)
- perform normalization using EdgeR's TMM method
- use lncDIFF (https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-019-5926-4) package to select normalized genes with log 2 fold change >1 and BH FDR <0.05. This package should have better performance compared to other differential expression tools such as DESeq2, edgeR, etc.
Would this be a good way to perform DEA of lncRNAs?
Thanks!
•
written
by
bart •
1 answer
Statistical analysis of RNA-seq for lncRNAs is done the same as for any other RNA-seq analysis and there isn't any particular need for new pipelines. I strongly recommend using a regular RNA-seq pipeline with read counts instead of RKPMs. You can follow the regular edgeR documentation. edgeR is quite capable of using fractional counts, if that is what you have. I don't personally agree that lncDIFF has better performance than edgeR. I would tend to analyse the lncRNAs together with protein-coding genes in order to make global normalization methods like TMM more reliable.
Log in to answer this question.
-
Proteomics DEA
written by frarodmar17 •
-
Assign gene to peak in bulk ATAC-seq
written by Chris •
-
A recent approach to identify lncRNAs from a large list of differentially expressed genes
written by diego1530 •
-
expression trend during cancer progression
written by Amir.Asgary •
-
Correlating RNA-seq and ChIP-seq data? Concurrence frequencies as metric?
written by stellaparker •
-
Slingshot: how do you choose which type of dimensionality reduction to use it with?
written by Friederike
-
How to perform lncRNA differential gene expression analysis (unstranded)?
written by mmitra •
-
question about survival analysis
written by tujuchuanli
-
Question about normalization and differential expression analysis
written by kamel •
-
Differential Expression using Flux capacitor output
written by vrehaman •
