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Hi,
Has anyone observed very high polyclonality from Ion Proton after the Qubit assay being in the optimal range for templated ISPs? We have pools of amplicon libraries that have some complementary sequences and they are trying to figure out the sequence of a 6 to 7 bp region. So the complexity of these libraries are fairly low. In the past, these libraries worked fine after emulsion PCR and gave us very low polyclonality (<10%) but in the recent batch they gave us polyclonality is >35%! They confirmed that nothing was changed in the preparation method but we did see a 8-20% of the library was migrating higher than their expected peak size. We thought this meant that there was denaturing and partial reannealing going on that would result in polyclonal beads if there are two templates annealed together. So, we dentaured the libraries before putting in the Ion Chef and got optimal % of templated ISPs but after sequencing got even higher % polyclonality. Any ideas? Could it be an issue with the Ion Chef emulsion? My next step is to try a serial dilution of the denatured template and try emulsions again.
Thanks in advance for any help with this.
Has anyone observed very high polyclonality from Ion Proton after the Qubit assay being in the optimal range for templated ISPs? We have pools of amplicon libraries that have some complementary sequences and they are trying to figure out the sequence of a 6 to 7 bp region. So the complexity of these libraries are fairly low. In the past, these libraries worked fine after emulsion PCR and gave us very low polyclonality (<10%) but in the recent batch they gave us polyclonality is >35%! They confirmed that nothing was changed in the preparation method but we did see a 8-20% of the library was migrating higher than their expected peak size. We thought this meant that there was denaturing and partial reannealing going on that would result in polyclonal beads if there are two templates annealed together. So, we dentaured the libraries before putting in the Ion Chef and got optimal % of templated ISPs but after sequencing got even higher % polyclonality. Any ideas? Could it be an issue with the Ion Chef emulsion? My next step is to try a serial dilution of the denatured template and try emulsions again.
Thanks in advance for any help with this.