Doubt about RNASeq Paired-end process
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12 months ago
Vicent • 0

Hi,

I have a doubt regarding how an RNASeq experiment is performed. For the sake of clarity, I'm gonna show a representation of a double stranded library fragment:

XXXXX-5'ACTGC-----------------------TTTTTT3'-OOOOO
XXXXX-3'TGACG-----------------------AAAAAA5'-OOOOO

My question is: When you load the single stranded DNA samples onto the flow cell for the RNASeq experiment, is there a way to control which samples are stuck to the oligos in the flow cell surface? I guess that my confusion stems from the paired-end sequencing process... Because how do you know if you are sequencing the forward or the reverse strand? Is there a way to only load the forward strand?

And furthermore, if each strand has both adapters, how do you control that the correct strand gets hybridised to the corresponding oligo in the correct direction, since both strands forward and reverse have both adapters?

I guess, what confuses me is that the forward strand has the adapter 1 (in my case, XXXXX) next to the 5' end, while the reverse strand has the adapter 2 (in my case, OOOOO) next to the 5' end. So both could be replicated if they get hybridised/stuck to the correct adapter in the correct direction, and then how do you know which is which?

Thanks

RNASEQ NGS Paired-end • 520 views
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This may be a question better suited to SeqAnswers

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Oh sorry, should I remove the post? Thanks

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