Bioanalyzer: What are these huge fragments (after Illumina TruSeq RNA sample prep)?

Sciurus · 12-10-2013, 05:51 AM

Hi everybody,

This has been my second time doing a library prep using total RNA from plants. I used the Illumina TruSeq RNA sample prep kit v2.
Last time, everything worked all fine but this time after the final pcr step, the bioanalyzer results all show a huge peak at the beginning of the curve (see attachment).

Can anybody tell me what this is, where it could have come from and what I could do next?

Thanks!!!

Mike2188 · 01-22-2014, 12:46 PM

Hey,

I am surprised nobody has answered this yet, but it looks like you overamplified your library. What happens is that you run out of sequencing primers, whether as a result of a) not enough sequencing primer, b) too much template added, or c) running an excessive amount of PCR cycles. When the reactions run out of primer, the template starts annealing to itself and forming these long concatemers. You should try redoing the library with a smaller template input and fewer PCR cycles.

Hope this helps!

Chipper · 01-22-2014, 02:10 PM

In the lane shown it looks more like ladder contamination or some weird adapter concatamers, if you were referring to the spikey patern in the long end.

ECO · 01-22-2014, 02:53 PM

Someone bumped the table during the run (spikes)

, and your library is overamplified...

Sciurus · 01-23-2014, 02:35 AM

Thanks, guys!
Yes, that's what we concluded in the end too - overamplification of the library due to too much starting material + too many pcr cycles.

I am redoing the library.

Mike2188 · 01-23-2014, 09:11 AM

Some people have sequenced overamplified libraries with success if redoing is expensive or not much of an option. Also, people have reannealed their libraries. What happens is the DNA forms ds products that aren't complementary near the middle so they run funny on the bioanalyzer or gels, but they still have relevant sequencing information.

Best of luck with the rest of your experiment!

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01-22-2014, 12:46 PM	#2
Mike2188 Member Location: Winnipeg Join Date: Oct 2013 Posts: 26	Hey, I am surprised nobody has answered this yet, but it looks like you overamplified your library. What happens is that you run out of sequencing primers, whether as a result of a) not enough sequencing primer, b) too much template added, or c) running an excessive amount of PCR cycles. When the reactions run out of primer, the template starts annealing to itself and forming these long concatemers. You should try redoing the library with a smaller template input and fewer PCR cycles. Hope this helps!

01-22-2014, 02:10 PM	#3
Chipper Senior Member Location: Sweden Join Date: Mar 2008 Posts: 324	In the lane shown it looks more like ladder contamination or some weird adapter concatamers, if you were referring to the spikey patern in the long end.

01-22-2014, 02:53 PM	#4
ECO --Site Admin-- Location: SF Bay Area, CA, USA Join Date: Oct 2007 Posts: 1,358	Someone bumped the table during the run (spikes) , and your library is overamplified...

01-23-2014, 02:35 AM	#5
Sciurus Member Location: Vancouver Join Date: Dec 2013 Posts: 23	Thanks, guys! Yes, that's what we concluded in the end too - overamplification of the library due to too much starting material + too many pcr cycles. I am redoing the library.

01-23-2014, 09:11 AM	#6
Mike2188 Member Location: Winnipeg Join Date: Oct 2013 Posts: 26	Some people have sequenced overamplified libraries with success if redoing is expensive or not much of an option. Also, people have reannealed their libraries. What happens is the DNA forms ds products that aren't complementary near the middle so they run funny on the bioanalyzer or gels, but they still have relevant sequencing information. Best of luck with the rest of your experiment!