I'm working with the Smartseq2 for Ribosome Profiling NGS library construction. The RNA template for this library for RT reaction is gel size selected and range from 20-41 bases alignment reads in previous sequencing data from my lab. Bioanalyzer always shows a single peak at ~170 bp.
I have purified library with three rounds of AMPure beads and now I'm facing contaminants of ~230 and ~300 bp in gel which can't be explained by my template size. I'm wondering if it's possible that template switching is taking place twice or even a third time after first 5' adapter synthesis by MMLV additional tail insertion in the 5' adapter cDNA. Thus these contaminants might be libraries with two and three 5' adapters added by multiple template switching. Is that possible?
I have purified library with three rounds of AMPure beads and now I'm facing contaminants of ~230 and ~300 bp in gel which can't be explained by my template size. I'm wondering if it's possible that template switching is taking place twice or even a third time after first 5' adapter synthesis by MMLV additional tail insertion in the 5' adapter cDNA. Thus these contaminants might be libraries with two and three 5' adapters added by multiple template switching. Is that possible?