Hi everyone!
I am doing Amplicon Sequencing on a 454 GS FLX Titanium Platform (so I am following the emPCR Method Manual – Lib-A SV) and I have a question regarding the calculation of the % bead enrichment
(on the protocol: 3.7 Sequencing Primer Annealing - step 6).
For some unexplained reason, the enrichment values are very high, from 40% to even 70% (except for only 3 samples which are around 20%). Do you think that something went wrong with the emulsions? Certainly they did not seem broken.
When I calculated the % Bead enrichment, for the total input beads I used as input value the number of recovered beads that I had previously counted from the % Bead Recovery (on the protocol: 3.6.1 Preparation for Indirect Enrichment - step 8), instead of the proposed by the protocol (4.0 × 10^6 for 1×Amp SVE) in order to be on the safe side. Do you think this is the correct way to calculate the % bead enrichment? I should mention that the % Bead Recovery rates were below 65% in most cases (which is considered typical) but there were around 62%.
The beads are counted with a Z1 Beckman Coulter and the DNA was quantified at a QuantiFluor, with the Quant-iT PicoGreen dsDNA Assay Kit, as proposed by Roche.
I am waiting for your suggestions!!
I am doing Amplicon Sequencing on a 454 GS FLX Titanium Platform (so I am following the emPCR Method Manual – Lib-A SV) and I have a question regarding the calculation of the % bead enrichment
(on the protocol: 3.7 Sequencing Primer Annealing - step 6).
For some unexplained reason, the enrichment values are very high, from 40% to even 70% (except for only 3 samples which are around 20%). Do you think that something went wrong with the emulsions? Certainly they did not seem broken.
When I calculated the % Bead enrichment, for the total input beads I used as input value the number of recovered beads that I had previously counted from the % Bead Recovery (on the protocol: 3.6.1 Preparation for Indirect Enrichment - step 8), instead of the proposed by the protocol (4.0 × 10^6 for 1×Amp SVE) in order to be on the safe side. Do you think this is the correct way to calculate the % bead enrichment? I should mention that the % Bead Recovery rates were below 65% in most cases (which is considered typical) but there were around 62%.
The beads are counted with a Z1 Beckman Coulter and the DNA was quantified at a QuantiFluor, with the Quant-iT PicoGreen dsDNA Assay Kit, as proposed by Roche.
I am waiting for your suggestions!!
Comment