GAGE analysis with the whole or a subset of matrix
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atakanekiz ▴ 30
@atakanekiz-15874
Last seen 6 months ago

Hello Bioconductor community,

I'm using GAGE package to analyze single cell RNAseq data, and I ran into a situation, I can't quite figure out. I tried finding out some explanation online, but I wasn't very successful.

I prepared an expression matrix from individual cells and annotated the column names to be able to subset on these cells(samples) in the GAGE analysis. I have multiple time points and genotypes and up to 14 clusters in my dataset. The names look like this: day9_wt_Act_CD8_1/2/3....   day12_ko_neutrophils_1.

I tried running GAGE analysis by using two different approaches:

1) Feeding the whole expression dataset into the function and selecting appropriate column indices for reference and sample comparisons (e.g. ref = d9_wt_act_cd8 vs ref= d9_ko_act_cd8). In this case, there are numerous columns which aren't used in comparisons. I ran the comparison "

2) Subsetting the matrix into only the samples that I'm interested in comparing. In this case, when I select the reference samples, the rest of the dataset is used in my comparisons, and there are no samples(columns) which isn't included in the comparisons.

Between these two approaches, I got quite different gene sets and statistics.  In the first approach (whole dataset as an input) comparing two subsets of data resulted in 8 genesets significantly (q<0.1) enriched. The second approach (trimmed expression matrix as an input to compare the same two subsets) resulted in 1 significantly enriched gene. I'm not sure which one to believe. Your insights are appreciated.

Thanks!

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moved my comment down as an answer

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atakanekiz ▴ 30
@atakanekiz-15874
Last seen 6 months ago

I actually figured out the problem. There was a mistake in my code while resulting in erroneous output. I can now confirm that regardless of the input matrix size, as long as the column annotations (ie ref vs samp are done properly, GAGE gives the same output. Hopefully, this might help somebody, someday.