I want to see differential expression, novel transcript, novel splice variants, and some information about NON coding RNA s too. Should I enrich my sample with poly Ts then, or just go for sequencing without any mRNA enrichment. Let me know the pros and cons of both of the strategies.
if you polyA+ select your RNA you're going to deplete for the ncRNA species. That doesn't mean you wont see them, I've seen miRNA and lncRNA in pA+ selected RNA, but I would have concerns over the accuracy of its quantification.
If you're using a model organism a RiboZero like approach might be better suited, preserving ncRNA in the sample. Be aware that you will have to sequence more in terms of raw read numbers to get the same sensitivity for alt-splicing etc. than if you had pA+ selected the RNA in the first place.
100M reads is enough to sequence without any prior enrichment procedure, which is, for sure, the best you could do about your sample. The less you modify your sample before sequencing the better. Important: think about replicates if you want to get reliable information about differential expression.
Assuming you're doing RiboZero with mammalian samples I agree with Alexander, 100M reads will be ample. Replicates means biological replicates. The general consensus would be that technical replicates are not required for RNA-Seq as the reproducibility is high between runs. However I would still split my samples between lanes.
For instance if you have 4 conditions and require 2 samples per lane for depth per condition (8 samples). I'd multiplex the sample 8 times across all four lanes, rather than putting experimental groupings into a single lane (condition 1 in lane 1, condition 2 in lane 2 etc,).
Thank you Daniel for this suggession. Yes, its highly doable. Thanks once again.
One more query I have, During Primary Data analysis should we consider removing duplicates ( SInce the fragmentation was done on the basis of Enzymatic Digestion). Please assist
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