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Question: RNA-seq time course experiment - LRT design hep
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gravatar for Maithê Barros
4 weeks ago by
Maithê Barros0 wrote:

Hello,

I am doing RNA-seq analysis of DE genes from mare's endometrial biopsies. We collected our samples at an abattoir due to restrictions with samples collected from live horses. 

Thus, I have three time points: alive_0h  (when the mare was just slaughtered, tissue representing the uterus of a live mare still). Then we took biopsies from the uterus and used them in a tissue culture. After culturing the explants we had 2 time points (24h and 48h) for both control and LPS challenge. 

I am having a hard time trying to figure out the design I should use since I have this samples at 0h. We have 5 biological replicates (horses). 

My LRT design is the following one:

ddsLRT_exp3 <- DESeqDataSet(se_exp3, design = ~ horse + treatment)

ddsLRT_exp3 <- estimateSizeFactors(ddsLRT_exp3)

ddsLRT_exp3 <- estimateDispersions(ddsLRT_exp3)

ddsLRT_exp3 <- nbinomLRT(ddsLRT_exp3, reduced = ~ horse)

resLRT_exp3 <- results(ddsLRT_exp3)

My colData is: 

samples,treatment,horse
sample_55.bam,alive_0h,7B
sample_56.bam,control_24h,7B
sample_57.bam,LPS_24h,7B
sample_58.bam,control_48h,7B
sample_59.bam,LPS_48h,7B
sample_60.bam,alive_0h,13D
sample_61.bam,control_24h,13D
sample_62.bam,LPS_24h,13D
sample_63.bam,control_48h,13D
sample_64.bam,LPS_48h,13D
sample_65.bam,alive_0h,14B
sample_66.bam,control_24h,14B
sample_67.bam,LPS_24h,14B
sample_68.bam,control_48h,14B
sample_69.bam,LPS_48h,14B
sample_70.bam,alive_0h,15A
sample_71.bam,control_24h,15A
sample_72.bam,LPS_24h,15A
sample_73.bam,control_48h,15A
sample_74.bam,LPS_48h,15A
sample_75.bam,alive_0h,15C
sample_76.bam,control_24h,15C
sample_77.bam,LPS_24h,15C
sample_78.bam,control_48h,15C
sample_79.bam,LPS_48h,15C

 

Our aims are to compare:

1)  Control vs LPS at 24h and 48h

2) LPS 24h vs 48h 

3) Alive 0h vs Control 24h vs Control 48h 

 

Many thanks indeed!

ADD COMMENTlink modified 4 weeks ago • written 4 weeks ago by Maithê Barros0

Thank you so much for the quick reply! I did think about it and I already ran something like that:

bam_exp3 <-c("sample_55.bam", "sample_56.bam", "sample_57.bam","sample_58.bam", "sample_59.bam", "sample_60.bam", "sample_61.bam", "sample_62.bam", "sample_63.bam", "sample_64.bam", "sample_65.bam", "sample_66.bam", "sample_67.bam", "sample_68.bam", "sample_69.bam", "sample_70.bam", "sample_71.bam", "sample_72.bam", "sample_73.bam", "sample_74.bam", "sample_75.bam", "sample_76.bam", "sample_77.bam", "sample_78.bam", "sample_79.bam")

library(Rsamtools)
bamfiles <- BamFileList(bam_exp3)

library(GenomicFeatures)
txdb_exp3 <- makeTxDbFromGFF("Equus_caballus.EquCab2.89.gtf", format="gtf")ebg_exp3 <- exonsBy(txdb_exp3, by="gene")

se_exp3 <- summarizeOverlaps(features=ebg_exp3, reads=bamfiles,
                            mode="Union",
                            singleEnd=FALSE,
                            ignore.strand=TRUE,
                            fragments=TRUE )
dim(se_exp3)
assayNames(se_exp3)

colSums(assay(se_exp3))

rowRanges(se_exp3)

sampleTableExp3 <- read.csv("exp3_metadata.csv")

colData(se_exp3) <- DataFrame(sampleTableExp3)

library(DESeq2)

dds_exp3 <- DESeqDataSet(se_exp3, design = ~ horse + treatment)
dds_exp3 <- DESeq(dds_exp3)

resultsNames(dds_exp3)
plotDispEsts(dds_exp3, main="Estimation of dispersion", sub="For DESeq2 object")

res_exp3_control24h_LPS24h <- results(dds_exp3, contrast=c("treatment", "control_24h", "LPS_24h"))
res_exp3_LPS24h_LPS48h <- results(dds_exp3, contrast=c("treatment", "LPS_24h", "LPS_48h"))
res_exp3_control48h_LPS48h <- results(dds_exp3, contrast=c("treatment", "control_48h", "LPS_48h"))

Would you give me your opinion? Does that look correct to you?

ADD REPLYlink written 4 weeks ago by Maithê Barros0
1

I moved this from an Answer to a Comment.

Yes that’s what I meant by combining variables and using contrast.

ADD REPLYlink written 4 weeks ago by Michael Love19k

Awesome! Thank you ever so much for your quick reply and for helping me out, Michael! 

ADD REPLYlink written 4 weeks ago by Maithê Barros0
1
gravatar for Michael Love
4 weeks ago by
Michael Love19k
United States
Michael Love19k wrote:

It sounds like you could accomplish your desired comparisons by combing time and condition into a single factor (see vignette section on interactions). So then you’d use ~horse + group, and make pairwise comparisons with results() and contrast.

ADD COMMENTlink modified 4 weeks ago • written 4 weeks ago by Michael Love19k
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