Calculation of % Bead enrichment (454 FLX Titanium)

[Christina85]

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!!

[Christina85]

I forgot to mention that I have performed the emulsion titration with 2 x 10^6 Capture Beads per emulsion tube and 4 x 10^6 Capture Beads per emulsion tube, to check which one will yield better % Bead enrichment, but there were barely any differences between the different emulsions.

[RCJK]

Hi Christina,

What cpb values did you use for your inputs? Have you tried repeating the reactions with lower cpb values? In my experience, I generally have to use a lower input amount for amplicon projects. I would also say to closely check for any broken or slightly broken emulsions. Those would be the two factors that would most likely result in such high enrichments. Hope this helps!

[Christina85]

I used 2 cpbs and 4 cpbs. 2 cpbs seemed to have worked just fine the previous times I run FLX (for 16S amplicons).
This time, I was not certain for the optimal cpbs because I am sequencing a different gene, so I decided to do titration for 2 and 4 cpbs.
I think I might try lowering the cpbs, even though 2 is the lowest Roche proposes.
The emulsion didn't seem broken, but based on the posts I 've checked so far they might have just as well been broken without any obvious change.

[ajthomas]

You might also have a contamination problem. If any of the components of the emPCR or the workspace where you set it up have been contaminated with the waste from a previous emPCR (especially from the emulsion breaking or first melt), you will get high enrichment values that don't seem to change much with different cpb ratios.

[pmiguel]

I used 2 cpbs and 4 cpbs. 2 cpbs seemed to have worked just fine the previous times I run FLX (for 16S amplicons).
This time, I was not certain for the optimal cpbs because I am sequencing a different gene, so I decided to do titration for 2 and 4 cpbs.

What is the typical ratio of beads to micro reactors? Anyone? That is, you add your template to the aqueous phase of a cocktail that becomes all the non-mock micro reactors. What percentage of those actually have a bead in them?

If every non-mock reactor had a bead in it, then you would want 0.1 to 0.2 cpb -- to get that 10 -20% enrichment value. But if most of the non-mock reactors are empty, that would explain why a higher ratio of template molecules to beads is needed.

--
Phillip