Too low enrichment

[JoanaIEE]

High everybody

I am new to this community, it is great to have such a platform to ask questions!

My problem is the following:
I have performed a successful 454 Junior Run with a Rapid Library kit and Lib-L emPCR. The sample consists of PCR products and I calculated with 2 fragments/bead for the emPCR. It worked fine only that I had a quite many fragments which were concatenated.
Therefore I made another Run with 1.5 ul of adapters instead of 1 to increase the adapterNA ratio. I used a very similar sample and the same library prep kit but I got almost no enriched DNA beads. I repeated the emPCR but again only very few enriched beads. Then I redid the library and again the same problem. Afterwards I took 5 times the amount of library and I got more enriched beads but still not enough for sequencing. So I did another emPCR with 80 fragments/bead and I got just enough DNA beads. I got about 60'000 good reads which is much less than I got with the first run but the quality was good and the fragments fully sequenced.
As I had increased the AMPure bead to DNA ratio to not loose my shorter fragments, I thought the problem was that I had adapters in the library which lead to a too high measurement of concentration.
But now I made a new library with a size cutoff at 400 bp (seen on the Bioananalyzer) and again I had only about 10-20% of the enriched beads needed after emPCR.

The emulsions were not broken and the PCR machine, NaOH solution and all lab equipment was successfully used for another sequencing run in the meantime.

I wonder if the adapter ligation may not have worked properly so that only few fragments have both adapters. Is there any way I could check that?
Or do you have any other suggestions?

Thank you.
Joana

[madseq]

Hi Joana,

you can check your library doing qPCR, we normally use the KAPA kit and works pretty well. It is normal to obtain 10 times less library molecules using this method, this is because you are only considering productive amplifiable molecules. You can use other LibL libraries that you have already sequenced so that you can establish the "ideal" number of molecules per bead for a successful run for your lab. In our lab, this number is about 0,17 qPCR dsDNA molecules/bead.

Nevertheless, 80 molecules/bead sounds too high to me. It doesn´t make sense. Make sure you use fresh melting solution every time.
Also, do you denature the library at 95ºC, 2 min right before adding it to the capture white beads? This is one of the major changes between LibA and LibL. I think that Junior users usually do amplicon sequencing, so you may have skipped this step?

[JoanaIEE]

Hi madseq
Thanks for your reply. I will certainly check out the Kapa kit.
I just don't understand why the first library worked perfectly with 2 CPB and for the second run I had to take 80 CPB.
I used fresh melting solution and even made a new 10 N NaOH solution and denaturated my library just before adding it to the capture beads.