bimodal distribution peaks observed in library prep

[Duuluu]

Hello everyone,

I am seeing two peaks (bimodal distribution) in my lib prep BioAnalyzer. The input sample is gDNA extracted from whole blood. Does anyone what this might be coming from, it looks like a shoulder peak. Could it be due to unoptimized PCR cycles?

Thanks

[Ben3]

Duuluu I have a few questions to help figure this out.

1. What library prep kit did you use? And did you make any changes to it?

2. How many PCR cycles did you use?

I would guess that your first assumption on the unoptimized PCR is the culprit, but I'm not completely sure.

[Duuluu]

Hi Ben3,

i used NEXTFLEX XP kit with no kit modification. Had 50ng dsDNA input and used 5 cycles of PCR. I plan on using 4 cycles instead of 5, and have more uniform fragmentation. Still not sure what caused it though.

[Ben3]

Interesting because that isn't a lot of cycles. I have had this happen before with another kit many years ago and I'm pretty sure the vendor said I should just lower the PCR cycles. But I also believe that when I sequenced those samples that I still had good data.

[Duuluu]

Ben3 so turns out the fragmentation needed to be longer. I was able to get much better data with proper fragmentation.

[Ben3]

Duuluu I'm glad it worked. What did you use for your fragmentation?