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-   -   Bioanalyzer: What are these huge fragments (after Illumina TruSeq RNA sample prep)? (http://seqanswers.com/forums/showthread.php?t=39109)

Sciurus 12-10-2013 04:51 AM

Bioanalyzer: What are these huge fragments (after Illumina TruSeq RNA sample prep)?
 
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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 11:46 AM

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 01:10 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 01:53 PM

Someone bumped the table during the run (spikes) ;), and your library is overamplified...

Sciurus 01-23-2014 01:35 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 08:11 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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