I sent off my first ddRAD library for sequencing and as QC it was first run through the bioanalyzer. I size selected with a pippin prep between 375-475 bp and the results of the bionalazyer show a very large peak at around 260 and a very low, low peak at around 438. I did not proceed with sequencing as all of my reads would be from the shorter peak. I was given the option to run my library through a pippin once again but I decided against that as I believe the final concentration of my target peak would be too low to sequence.
I'm trying to think through what may have gone wrong. I'm thinking there was most likely an amplification issue at the PCR stage. Any ideas? I can provide more info if needed!
Hi Valentina,
The shorter peaks can be from two reasons:
1. It can be of adapters (including indexes), or
2. During PCR most mostly shorter fragments got amplified (We know that the PCR is biased towards shorter amplicons)
The one thing I am not able to understand, why are you seeing this patter after size selection. It might also be a problem with your pippin prep cassette or some error during the size selection.
I recommend if this happens again, do the size selection using Ampure XP beads instead of pippin.
- Badges
- Science topic
- Similar topics
- Biological Science
- Kinesiology
- Running