Not sure of an answer, but for seq-releated questions I recommend the forums: seqanswers or biostars. 

https://www.biostars.org/

http://seqanswers.com/

Miriam,

this is first of all a question of money. For sure its better to have individual samples in order to see variances what you cannot do in pooled samples. The question is whether you want to do a hypothesis-generating experiment or not. For the generation of hypotheses pooled samples should be okay.

Hi Miriam

I think a single barcoded  sample will be ideal to see transcript variations and expressions.

But, If you would like to save some money then that would work, remember it will not be ideal technically especially demultiplexing of transcripts (in order to study individual samples) within the other samples will be very difficult

Thank you all for the info! I belive that maybe sequencing several samples in the same lane and with different index would be a better option as, although compromising coverage, I can have more samples sequenced per group.

Hi Miriam,

Pooling RNA from many samples of one comparison group before sequencing reduces the cost and theoretically should work better because you obtain average level of expression across all samples. While in practice, there is a risk to bias an experiment. Imagine you have an outlier among your pooled samples. This outlier will drag the average expression values towards its values and thus bias your results. On the other hand, sequencing of each samples separately enables you too check all samples and identify any outlying samples. But it will cost more.

Pooling several samples together is also tricky because it is difficult to assure that all samples have exactly the same total amount of RNA and contribute equally to the pool.

I have read a paper discussing all these aspects a year ago when I planned my RNA-Seq experiment. I cannot find it right now though. If I find it I will post it here.

So, it is up to you. But it is usually recommended to sequence each sample separately, even if you sequence smaller number of samples.

No. Avoid pooling whenever you may. You may loose signal for important genes. 

Pooling was done in the old times, when cost of RNAseq were prohibitive. Or when you surely do not have enough material (embryos). 

I would like to recommend the following article about experimental design in RNA-seq.

Paul L. Auer and R. W. Doerge. Statistical Design and Analysis of RNA Sequencing Data. Genetics. 2010 Jun; 185(2): 405–416.

I am strongly convinced that good experimental design can significantly reduce biases in gene expression analysis. I suggest to have at least two biological and two technical replicates for each condition. Thus, a broader range of statistical tools can be applied for conducting differential gene expression analysis.    

I would not recommend pooling either. If you know people who already did RNA-seq on pig samples or if the sequencing facility you're going to work with has experience in RNA-seq in pigs, you could discuss with them and adapt the sequencing depth : go with few biological replicates with a high coverage or with more replicates with a lower coverage.

You can also look at this article

Rajkumar et al. 2015. Experimental validation of methods for differential gene expression analysis and sample pooling in RNA-seq. BMC Genomics 16:548. DOI 10.1186/s12864-015-1767-y

Best,

Thank you all for your help, I am definitely going to go for the individual sample approach, although reducing the sequencing depth.

Thanks!

I think that's a wise idea, since I was about to answer the same as the previous people. However, depending on your sequencer, you can now multiplex quite a lot of samples by lane. A typical HiSeq 1500 lane provides about 180 million reads, which should be enough for 4 to 6 samples, so between 32 and 48 per flow cell, for a cost of about 400 € per sample, library prep included (external provider). Good luck!

You may want to read our recent paper Article On the utility of RNA sample pooling to optimize cost and st...

try to avoid pooling as much as you can, sequencing cost will be reduced when you do more samples (negotiate with the company that is doing the sequencing for you to reduce the cost, if not doing it yourself). best wishes