Hi
I have 20 RNA samples for miRNA sequencing because of its high cost, Some colleagues suggested pooling the samples, it means, splashing the samples into one or two sample and send for sequencing.
Is it a regular way because I haven't found publication yet?
Some also set up barcodes before or after the library is set up, which is more expensive.
Thanks for your tips
There is this paper (https://www.pnas.org/content/pnas/102/12/4252.full.pdf). It discusses and evaluates the concept of pooling in microarray, but I think some of the results apply to sequencing data as well.
My own view is that the variance you get from pooled samples is the technical variance. This is because the biological variability between the samples would likely be averaged. So depending on the purpose of the experiment that could be fine or even desired.
As a side, one or two samples are never enough to extract reliable signals. You can pool the 20 samples into one pool and then sequence it enough times to be able to detect signal.
Hi Ali
pooling samples for NGS study will give you an average of all your targets in only one sample, erasing the diversity of your samples. The best way is to prepare separately all samples for library and after barcoding you can cheer pool all librairies in one sample to be sequenced. It's cheaper and you'll keep samples diversity. ok there are some bias in barcoding mainly due to sample quality and therefore you'll need to have a look on qualification, as in all NGS purposes. I agree with Mahmoud Ahmed that miRNA are poorly expressed in some tissues but the way you prepare your samples is the way you'll get data and be able to analyse them.
fred
Ali Mahmoudi
Pooling is dependent on the research question or objectives you want to achieve. If all you need is enough RNA from particular samples, its okay to make a pool and sequence. But if you want to relate the RNA sequence data to particular samples then you have to invest in labelling/indexing each sample before you make a pool.
Hope this is helpful,
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