beads post e-80 enrichment

[jayalakshmi]

Hi all,
I had done a e-PCR enrichment e-80 scale for a whole transcriptome lib (8 samples pooled). Post enrichment I had got a yield of around 3 billion (which is way too higher than the expected yield of around 1.5 to 2 billion for an e-80 scale). The titration point used for the e-PCR was 0.6pM (protocol recommends between 0.4-0- 0.8pM). Post loading on the flowchip and after the first bead assesment report, the % on axis also is on the lower side(58%, the dots on the satay appear to be scattered as opposed to being tightly clusterd around the axis, refer the attached pic).
While monitoring the run in real time the height of the bars also keeps decreasing post each ligation(refer the pic) and the % of good +best beads also falls till 20% (it started at 60%).

Has anybody experienced a problem of getting a higher amount of beads than expected during the enrichment process ? What could be reason for that? would decreasing the titration point solve the issue, since for WT the library size is broad?

Kindly advice.
Thanks,
Regards,
Jaya

[JackQuist]

Hey Jaya,

This is a rather low quality run. There are a number of factors which to cause a drop in G+B beads and high bead yield.

First: is your EZ Bead up to date? There are a number of software and hardware fixes that were released post launch. Current is version EZ1.9. Email: [email protected] for the upgrade patch (free). If you have an original you are running version 1.6!

Now about the high yield...it's on the high side but E80s can hit 3 B beads. However you also note that the satay shows off axis beads. That's a key indicator your DNA library titration point is probably off. The result of too much library being added into the emulsion would be exactly what you see...a high bead yield and off-axis beads and a steep drop in good plus best. Suggest re-calibrating your DNA curve. Alternative is just empirically trying a lower titration point but know that your DNA curve is likely off.

Other possibilities for your problem include mechanical failures on the Enricher.

Best of luck.

[JPC]

Hi jayalakshmi

we've seen this problem before and traced the issue back to a problem with quantification, what method do you use?

Too much input DNA leads to polyclonal beads on the slide which will give you your poor satays as polyclonal beads will often have two colours following a ligation.

We've been told to ignore the green bars unless they stop being green. we don't see a correlation between hight and quality.