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Hello all
I have been working with microbiome libraries using Illumina's recommended protocol and sequencing the V3-V4 regions. My question is about the post-indexed library concentrations. I am getting wildly variable concentrations of the indexed samples (measured by qubit, ranging from too low to be detected to 38 ng/uL) with the expected size (~630 bp). I'm just curious what other people typically see for their concentrations at this step. We are following the Illumina protocol and using 8 cycles of their recommended conditions. Should we increase the number of cycles at this step to increase our concentrations? What about increasing the amount of starting template from 5 uL to 10 uL or 15 uL? Any advice is greatly appreciated.
I have been working with microbiome libraries using Illumina's recommended protocol and sequencing the V3-V4 regions. My question is about the post-indexed library concentrations. I am getting wildly variable concentrations of the indexed samples (measured by qubit, ranging from too low to be detected to 38 ng/uL) with the expected size (~630 bp). I'm just curious what other people typically see for their concentrations at this step. We are following the Illumina protocol and using 8 cycles of their recommended conditions. Should we increase the number of cycles at this step to increase our concentrations? What about increasing the amount of starting template from 5 uL to 10 uL or 15 uL? Any advice is greatly appreciated.