Question: How is it possible to recognize replicates?
0
gravatar for Maya
7 weeks ago by
Maya0
Maya0 wrote:

Hi to all,

I'm new in analyzing RNA-seq data. I have to evaluate gene expression in different cells and tissues available on online datasets. To perform DEA I need replicates of course. How can I be sure that these below are technical replicates?

enter image description here

They derive from the same experiment, I mean same protocol, same day of library preparation, same day of sequencing and same sequencing technology but they are different runs. In your opinion are they replicates?

Thank you in advance for your time and suggestions!

rna-seq • 157 views
ADD COMMENTlink modified 7 weeks ago by Carlo Yague5.5k • written 7 weeks ago by Maya0
1
gravatar for Carlo Yague
7 weeks ago by
Carlo Yague5.5k
Canada
Carlo Yague5.5k wrote:

These loos like these technical duplicates indeed. According to "library name" field under the ERR258415[0-1] run pages, both datasets are independant runs on the same sRNA library (called 683610_Occipital_Cortex).

But it might be best to contact the authors directly if you want to be 100% sure.

ADD COMMENTlink written 7 weeks ago by Carlo Yague5.5k

Ok thank you, it is also my idea. Probably I will contact them. I have also another question. From a biological point of view does it make sense to compare tissues from different experiments (same protocol of RNA-extraction and technology of sequencing but different day of library preparation and of sequencing) or the risk of batch effect is too high?

ADD REPLYlink written 7 weeks ago by Maya0

There will be a batch effect for sure ! Sometimes it is small, sometimes big, but it is best to avoid it OR control for it (for instance, resequencing a bit of the first libraries along the newer samples).

ADD REPLYlink written 7 weeks ago by Carlo Yague5.5k

So the best option is to compare tissues or cells processed the same day to avoid batch effect (of course we cannot exclude it also in this case), right ? Instead to compare tissues or cells processed in different days can I proceed with an approach to estimate the batch effect? Actually I read different questions and posts about how to remove batch effect for example with PCA or edgeR

ADD REPLYlink written 7 weeks ago by Maya0

So the best option is to compare tissues or cells processed the same day to avoid batch effect ,

Yes

Instead to compare tissues or cells processed in different days can I proceed with an approach to estimate the batch effect?

Yes, this would be the second best option. But to estimate the batch effect, you need at least one sample sequenced across batches. Then it is possible to remove/control the batch effect. using edgeR, DESeq2, or something else.

ADD REPLYlink written 7 weeks ago by Carlo Yague5.5k

Thank you for your explanation, always very clear. What do you think about this bioconductor package http://bioconductor.org/packages/release/bioc/html/debrowser.html for less expert users? Have you ever heard about this?

ADD REPLYlink modified 7 weeks ago • written 7 weeks ago by Maya0

I do not know that package, sorry

ADD REPLYlink written 7 weeks ago by Carlo Yague5.5k

Ok, thank you again for your clear answers!

ADD REPLYlink written 7 weeks ago by Maya0

if that was useful, you can accept my answer above :)

ADD REPLYlink written 7 weeks ago by Carlo Yague5.5k
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