Hello! First time poster, although have lurked for a while.
I have looked through the site for advice on library quantification, and understand that ideally a combination of Qubit, Bioanalyser and qPCR should be used.
However, I am trying to use the MiSeq to generate reads out of transposons into neighbouring genome (TraDIS), and i'm not sure if the same constraints apply to my library prep in comparison to the majority of other applications.
Firstly, in TraDIS the number of reads is more important (to an extent) than the length of the reads. As such, is tightly defining the fragment length of my library as important as it is for other applications? Does having a wider range of fragment lengths on the flow cell affect cluster generation/PF/read quality? An important consideration here is that I would like to avoid any bias introduction through stringent size selection.
Secondly, assuming a wider fragment range in the library, would qPCR be the best option for quantification? Correlating Qubit concentrations and Bioanalyser traces becomes more problematic with a wider range library; would qPCR totally solve this issue?
As may be evident, I am still working through the library prep to finalise a standardised, reliable protocol. There are other aspects I need to look into, but I think it makes sense to iron out the basis of experiment. Any and all opinions are appreciated, and apologies for the long post!
I have looked through the site for advice on library quantification, and understand that ideally a combination of Qubit, Bioanalyser and qPCR should be used.
However, I am trying to use the MiSeq to generate reads out of transposons into neighbouring genome (TraDIS), and i'm not sure if the same constraints apply to my library prep in comparison to the majority of other applications.
Firstly, in TraDIS the number of reads is more important (to an extent) than the length of the reads. As such, is tightly defining the fragment length of my library as important as it is for other applications? Does having a wider range of fragment lengths on the flow cell affect cluster generation/PF/read quality? An important consideration here is that I would like to avoid any bias introduction through stringent size selection.
Secondly, assuming a wider fragment range in the library, would qPCR be the best option for quantification? Correlating Qubit concentrations and Bioanalyser traces becomes more problematic with a wider range library; would qPCR totally solve this issue?
As may be evident, I am still working through the library prep to finalise a standardised, reliable protocol. There are other aspects I need to look into, but I think it makes sense to iron out the basis of experiment. Any and all opinions are appreciated, and apologies for the long post!
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