Hello,
I’m about starting a sequencing experiment with a HiScan SQ.
The point is that we have to prepare the library from multiplexed fragments, varying from 200 to 800 bp, but we are trying to find out how to have a final DNA input where all the amplicons got an equimolecular representation in order to provide a good number of reads and coverage at the end.
Does anyone know how to normalize the effect of mixes with different number of amplicons and concentration? Or this isn’t that important as far as you keep your samples in a good average amplified range?
We tried to get them at the same concentration but it has been impossible. We will have to amplify some of them even in uniplex.
Our aim is to identify SNPs and we are afraid we introduce a bias already before starting the library prep that could decrease the final quality during the analysis.
We usually work with NEBnext and TrueSeq DNA kits and fragment by Covaris method.
I would appreciate very much every help or experience you could provide.
Best regards,
Xavi
I’m about starting a sequencing experiment with a HiScan SQ.
The point is that we have to prepare the library from multiplexed fragments, varying from 200 to 800 bp, but we are trying to find out how to have a final DNA input where all the amplicons got an equimolecular representation in order to provide a good number of reads and coverage at the end.
Does anyone know how to normalize the effect of mixes with different number of amplicons and concentration? Or this isn’t that important as far as you keep your samples in a good average amplified range?
We tried to get them at the same concentration but it has been impossible. We will have to amplify some of them even in uniplex.
Our aim is to identify SNPs and we are afraid we introduce a bias already before starting the library prep that could decrease the final quality during the analysis.
We usually work with NEBnext and TrueSeq DNA kits and fragment by Covaris method.
I would appreciate very much every help or experience you could provide.
Best regards,
Xavi
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