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Old 01-23-2018, 12:52 AM   #1
mengal
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Default Biological replicates

Hello everyone,

I am working on infertility of male cattleyak, RNA-seq data. I have cattle fertile and cattleyak infertile epididymal tissue samples.I have sent the tissue sample to a company for RNA seq (C1, C2, C3- Y1, Y2, Y3) but the company couldn't,t get the good quality RNA for C1 biological replicate and the excluded that so now the results for comparison I have is like this (C2, C3- Y1, Y2, Y3) is that okay to processed with results


-Can anybody tell me what to do??

Please help & tell me how should I proceed.
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Old 01-23-2018, 01:04 PM   #2
sdriscoll
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What you're probably concerned with is if you can rely on differential expression results from a small sample size such as 2 vs 3. The differential expression tools (DESeq2, edgeR, etc..) have very low false positive rates even with small sample sizes. Unfortunately they also likely have pretty high false negative rates at low sample sizes. So you're far more likely to be missing significant hits than getting false hits. This is true of even 5 vs 5 or 10 vs 10. They statistical analysis has been designed to have a low false positive rate in all cases.

In short, I'd say if you were planning to have a 3 vs 3 and now you have a 2 vs 3 you're not losing much. But you really don't know for certain until you see the data. Sometimes we sequence something we haven't sequenced before and it turns out that it's far more variable from sample to sample than other things and so our usual 3 biological replicates isn't sufficient anymore. I'd say you're gonna be OK and any positive hits you get back from a differential test should be reliable.
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Old 01-24-2018, 12:42 AM   #3
mengal
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bundle of thanks for your kind reply
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Old 01-24-2018, 12:51 AM   #4
mengal
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kindly tell me what to do next the company has done the analysis in two ways (M1, M2, M3, VS P2, P3) And (M1, M2, VS P2, P3) and there is a very big difference in DEGs. for the first combination the number of DEGs is (3008) and for the second one it's(6712) this is the url link for the Pearson's correlation coefficient r (Pearson's Correlation Coefficient) as biological repeat correlation assessment" and <a href="http://pt-br.tinypic.com?ref=10r95rb" target="_blank"><img src="http://i67.tinypic.com/10r95rb.jpg" border="0" alt="Image and video hosting by TinyPic"></a>" if you can kindly help cuz i'm a new researcher in this field it is very difficult for me take a decision.

thanks
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Old 01-26-2018, 10:19 AM   #5
torben
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Quote:
Originally Posted by mengal View Post
[T]he company has done the analysis in two ways (M1, M2, M3, VS P2, P3) And (M1, M2, VS P2, P3) and there is a very big difference in DEGs. for the first combination the number of DEGs is (3008) and for the second one it's(6712)
From the numbers I would assume that the second combination (M1, M2, VS P2, P3) was done as a paired test. However, from the description of your experimental setup there's no pairing so you should discard this analysis.
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Old 01-26-2018, 08:53 PM   #6
mengal
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Originally Posted by torben View Post
From the numbers I would assume that the second combination (M1, M2, VS P2, P3) was done as a paired test. However, from the description of your experimental setup, there's no pairing so you should discard this analysis.
Do you mean I should discard the second combination (M1, M2, VS P2, P3) and go with this combination (M1, M2, M3 VS P2, P3) ??

I really appreciate if you could kindly share your email ID. I really need your help because I Don't have a supervisor for this project and I'm confused with the results. this is my email id: [email protected]

thanks
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