Deseq2 Results are different using batch design
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@hemantcnaik-23771
Last seen 9 hours ago
India

Hello I am doing differential gene expression RNA seq data using DESeq2 data is sequenced using two different time different sequencing strategies I used with and without batch correction in my design as mentioned below

My design

Sample  Type    batch
Sample1 WT  1
Sample2 WT  1
Sample3 WT  1
Sample4 KO2 2
Sample5 KO2 2
Sample6 KO1 2
Sample7 KO1 2
Sample8 KO1 1


#with this my qRT-PCR results are matching 
dds <- DESeqDataSetFromMatrix(countData=countdata, colData=condition, design=~Type)
dds$Type <- relevel(dds$Type, ref = "WT")
dds <- DESeq(dds)
res <- results(dds)

#with this my qRT-PCR results are not matching 
dds <- DESeqDataSetFromMatrix(countData=countdata, colData=condition, design=~batch+Type)
dds$Type <- relevel(dds$Type, ref = "WT")
dds <- DESeq(dds)
res <- results(dds)

please help me anything I am missing here not able understand which data to consider

Thank you

DESeq2 • 712 views
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What is your question here ?

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My RNA data is sequenced by different time point as batch affect when I am including ~batch+type in my design matrix results were not matching with qRT results my genes of interest if I include ~type results are because of design two different way giving me different results which on is correct method and how to correct it?

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@mikelove
Last seen 4 hours ago
United States

This is entirely expected.

If you do not control for variables that influence the outcome (the counts), then the model is mis-specified and can lead to spurious results.

Generally we include all batch variables. If there are many, they are unknown, or correlated, we often recommend to use factor analysis methods. But here you would just include your known batch variable.

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I have edited my question batch variable I have added as sequence_by column which has 3 WT samples and 1 KO1 sequenced by different time point and another Two KO1 and KO2 sequence at another time point which I have to consider for batch or not? bit confused here

PCA without batch remove from limma

PCA after remove batch from limma

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While the design is nearly perfectly confounded, because of this group:

Sample6 KO1 2
Sample7 KO1 2
Sample8 KO1 1

You should be able to estimate both the batch and type effects.

It would have been better to distribute all types in all batches (achieving balance).

But you can still recover the comparison of interest if you include batch and type in the design.

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When I am include batch and type in my Design (~batch+Type) results are not matching with qRT this is my major concern for many genes (N=5) ~Type results are matching any comment on this ? Anything I have to change in my design matrix

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This is probably due to the confounding (all WT in batch 1, all K01 in batch 2).

It's not an ideal design, because you end up with a very noisy estimate of batch and condition effects (all relying on sample 8 to estimate the difference).

I don't have further recommendations on the statistical analysis plan, I've given all the information I can.

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Okay Thank you for your suggestion

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