Larger size TruSeq nano prep

[anna_m]

Hello,
I followed the protocol for the 550bp size TruSeq nano library preparation but my final libraries were much larger than the expected 670bp average size (see attached pic).

Has anyone else come across larger than expected libraries?
Do you have any advice as to what may have happened?

Thanks in advance,
Anna.

[sweetph3]

I also saw a larger than expected product. I ran the Covaris to shear at 500 bp, and the final libraries had Bioanalyzer peaks at 780 bp. I don't have an explanation.

[barrmur]

Truseq pcr free libraries do run larger on a BA due to the tailed adapters but I have not seen this before with nano libraries. One explanation my be incomplete mixing of the size selection beads meaning that the incorrect amount of beads were added to the cleanup and a poor size selection. Might also be an idea to run the sheared samples on the BA before cleanup to check the size distribution from the shearing.

[anna_m]

Hello, I asked Illumina tech support and they said to try diluting the libraries as the chip looked to be overloaded. I made a 1 in 20 dilution and repeated the chip. An example of a trace is attached. The average size has gone down by around 100bp but it's still not the 670bp average stated in the manual. I have sent tech support these diluted library results and asked if they think they will sequence.

Anna.

[jpmirandam]

I have the same problem. I make a change on the fragmentation protocol whit covaris (from a 550 pb insert to a 400 pb insert) but the mean size was almost the same (900 bp aprox.). I think that could be a problem on the size selection. Any help?. Thanks.

[anna_m]

Hello,
It's a size selection issue. The protocol doesn't need to be altered, just make sure your SP beads are vortexed a lot (I mean a lot!). I usually mix them in between each sample prep as a pre-caution. Also we've found that the libraries run larger on the tapestation in comparison to the bioanalyser. Libraries usually look around 50bp larger on the tapestation. When the insert sizes are checked in the analysis stage, they are usually around the right size.

All the best,
Anna.