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.
-
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.Leave a comment:
-
beads post e-80 enrichment
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
-
Data variability is still an issue in sequencing technologies despite the advances in reproducibility and accuracy of these platforms. But the problem does not originate in the sequencing itself, but in the previous steps, before the sample reaches the sequencer.
The first step is collection, followed by preservation and sample preparation for analysis. Most scientists overlook those steps, but not being careful might just be skewing the experiment’s results.
...Yesterday, 10:05 AM -
With the launch of new single-cell sequencing platforms in 2026, the field stands at an exciting inflection point. This article surveys the most impactful advances in the field and discusses how they’re reshaping research in cancer, immunology, and beyond.
Introduction
Single-cell sequencing technologies have undergone remarkable advances over the past decade, transitioning from low-throughput experimental approaches to highly scalable platforms capable of...05-22-2026, 06:42 AM -
Studying ecosystems means dealing with complex, multi-species communities that are hard to observe at scale. This complexity, however, hides many important questions to be answered, from how biogeochemical cycles work and how climate change can affect species distribution to how conservation strategies can work best.
Genomics, particularly since the expansion of NGS, has transformed ecosystem ecology. By sequencing environmental DNA, we can now assess biodiversity without direct...05-06-2026, 09:04 AM
Leave a comment: