Question: How to evaluate the similarity between two different samples by using RNA-Seq?
gravatar for dz2353
9 days ago by
dz235370 wrote:

Hi, there!

I have two samples' RNA-Seq data, one is amniotic epithelial cells(AEC), and another is keratinocyte(KRT). I have done with the upstream analysis and get the original reads count matrix. After that, I did PCA analysis, differential gene expression analysis by using DESeq2. Actually, I want to find out the similarity between AEC and KRT at the gene level. But I do not know how to do that cause I do not think the un-differently expressed genes from the result of DESeq2 can represent the similarity. I only have two samples and each one has one replicate, so I can not do co-expression network analysis. Anyone can help me? Thanks in advance!

rna-seq gene • 112 views
ADD COMMENTlink modified 7 days ago by Charles Warden6.6k • written 9 days ago by dz235370

I only have two samples and each one has one replicate...

Do you mean you have n=1 for each group? It is impossible to do proper statistics with such a 'poor' design (no offense), please consider adding more biological replicates.

Similarity between samples can be evaluated with clustering, such as hierarchical clustering. But also correlation can be used as a measure for similarity.

ADD REPLYlink written 9 days ago by Benn6.6k

Sorry, I mean each group has two replicates. A_1 and A_2 in AEC group, K_1 and K_2 in KRT group.

ADD REPLYlink written 9 days ago by dz235370

Are these technical replicates? For sound statistics you need biological reps... try to calculate correlation between your samples, and make a heatmap such as here. Correlation of 1 means similar, correlation of 0 means not similar.

ADD REPLYlink modified 9 days ago • written 9 days ago by Benn6.6k

Yes, they are biological replicates and I've done with correlation analysis. But actually what I want to do is to find out a gene list that shows the same expression level between two groups. Do you think the complementary set of differently expressed gene list is my target?Thanks for your reply!

ADD REPLYlink written 8 days ago by dz235370

Sounds like you are looking for equivalence test, haven't seen that before with RNA-seq data but if that's what you need maybe worth a try.

ADD REPLYlink written 8 days ago by Benn6.6k
gravatar for Charles Warden
7 days ago by
Charles Warden6.6k
Duarte, CA
Charles Warden6.6k wrote:

PCA and a dendrogram with hierarchical clustering (with Pearson Dissimilarity and/or Euclidan Distance as the distance metric) are the main things I would use to assess replicates before differential expression.

Otherwise, I would create a heatmap of differential expressed genes. Even if gene list sizes are similar, you may visually see better consistency of replicates with one method versus another (and I would test DESeq2/edgeR/limma-voom for your n=4 comparison).

ADD COMMENTlink written 7 days ago by Charles Warden6.6k

Thanks a lot, Charles.

ADD REPLYlink written 7 days ago by dz235370
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