Tweet
Using the illumina mRNA Seq kits I created 8 libraries last week; they are completed through pcr enrichment and ready to run on the GAs. For the most part the traces looked good however on all 8 samples there is an odd peak at around 75 bps. When I performed a gel cut out at ~300 bps I don't believe I introduced any other material so I am not sure where the extra peak is coming from. It seems to me that it could be a primer dimer from pcr enrichment or some sort of contamination in the gel of my bioanalyzer kit, either way I don't think those would affect the reads on a GA but I'd like to hear some input from you guys. The scans are attached bellow
-
-
If you used a TruSeq kit to construct these, then my guess is that the ~75 "bps" peak is actually the PCR primers used for enrichment PCR. These primers, from the "PPC" (PCR primer cocktail) tube give a peak in that region of the electropherogram of both DNA high sensitivity chips and RNA (pico) chips. See:
We don't run the DNA1000 chips in our lab, so have never looked at it on that type of chip.
--
Phillip
-
Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
-
I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
Comment