sharp peak in library - TruSeq Stranded Total RNA (rRNA-depleted E. coli)

Seqasaurus · 03-17-2015, 07:13 AM

Anyone seen anything like the sharp peaks at 250 bp in the bioanalyzer traces of the libraries below? Overamplified? Bubble product?

These are from rRNA depleted (Ribo zero) total RNA from E. coli. Prepared with a TruSeq Stranded Total RNA kit with the default fragmentation conditions.

Pab77 · 11-05-2015, 02:02 AM

The hump after 400bp could be a bubble product but the sharp peak at 250bp could be a population of RNA around ~125bp that is inherent to this sample or a contaminant of some sort. Remember that short fragments preferentially amplify in the PCR. I saw this type of peak once when I added too much spike-in RNA by mistake and it amplified much better than the sample RNA. Also this population will cluster better too taking more reads. If you didn't expect it, best to repeat library prep and check after rRNA depletion step. We routinely elute in water after RNA Clean XP clean up and run on the bioanalyzer to make sure rRNA is mostly depleted and how the profile looks.

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11-05-2015, 02:02 AM	#2
Pab77 Junior Member Location: Seattle Join Date: Feb 2012 Posts: 9	The hump after 400bp could be a bubble product but the sharp peak at 250bp could be a population of RNA around ~125bp that is inherent to this sample or a contaminant of some sort. Remember that short fragments preferentially amplify in the PCR. I saw this type of peak once when I added too much spike-in RNA by mistake and it amplified much better than the sample RNA. Also this population will cluster better too taking more reads. If you didn't expect it, best to repeat library prep and check after rRNA depletion step. We routinely elute in water after RNA Clean XP clean up and run on the bioanalyzer to make sure rRNA is mostly depleted and how the profile looks.