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-   -   ddRAD Post-PCR Bioanalyzer Has Multiple Peaks (http://seqanswers.com/forums/showthread.php?t=79740)

evobio721 12-21-2017 10:27 AM

ddRAD Post-PCR Bioanalyzer Has Multiple Peaks
 
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Hello,

I have a problem with a post-PCR ddRAD library that was prepared using Peterson (2012). It was prepared along with another index for sequencing on one Illumina lane, and the pre-PCR size selection for both indices was supposed to be 454-509 bp.

However, the Bioanalyzer results show two smaller peaks for one of the indices that shouldn't be there (see attached images), and the smaller peaks should be outside the size-selection range. Thus, it must have post size selection.

Does anyone know what might have caused the two smaller peaks to occur, and should I worry about it during sequencing?

Thank you,

-Bradley

BioKiwi 12-21-2017 11:26 AM

I wonder how many PCR cycles you have done and what was the total yield.

evobio721 12-21-2017 11:31 AM

Thank you for your prompt reply. I did 12 PCR cycles. The post-PCR yield was 45.6 ng/uL, which come to think of it does seem high. Pre-PCR DNA was ~1 ng/uL.

nucacidhunter 12-21-2017 01:57 PM

If you have run size selected tags on BA or still have remaining to run you can check for the presence of those peaks. I think the size selection has been sub optimal.

Other weak possibility is over amplification. I wonder what was the PCR input and final library elution volumes.

If it turns out to be over-amplification then a new library needs to be prepped. If it is result of size selection then you just will loose some reads and library can be sequenced.

evobio721 12-21-2017 02:09 PM

Quote:

Originally Posted by nucacidhunter (Post 213570)
If you have run size selected tags on BA or still have remaining to run you can check for the presence of those peaks. I think the size selection has been sub optimal.

Other weak possibility is over amplification. I wonder what was the PCR input and final library elution volumes.

If it turns out to be over-amplification then a new library needs to be prepped. If it is result of size selection then you just will loose some reads and library can be sequenced.

Thanks for your input. The size selection was done on a Pippin Prep, and the elution volume was what the Pippin Prep manual recommends (can't remember exact amount at the moment, but I think 40uL?). PCR input was 5 uL repeated four times, followed by post-PCR AMPure XP pooling/cleanup of PCR product.

That's a good idea about running the size selected DNA on the BA. I will try that to see if the issue is pre or post PCR. I imagine that's about the only thing I can do at this point outside of re-doing library prep.

nucacidhunter 12-21-2017 02:33 PM

So you have used in total 20 ng of size selected tags for PCRs and got 45.6 ng/ul after pooled PCR clean up. I am interested to know what was the elution volume from beads to look at possibility of over-amplification.

evobio721 12-21-2017 02:42 PM

Quote:

Originally Posted by nucacidhunter (Post 213572)
So you have used in total 20 ng of size selected tags for PCRs and got 45.6 ng/ul after pooled PCR clean up. I am interested to know what was the elution volume from beads to look at possibility of over-amplification.

After the post-PCR AMPure cleanup, I eluted in 30 uL. So there was 45.6 ng/uL in 30 uL.

I should note that when I did the PCR, both the index that worked well (first image) and the index that contained the multiple peaks were done at the same time.

nucacidhunter 12-21-2017 03:14 PM

It does not seem to be result of over-amplification. Most likely cassette lane has been faulty or other weak cause could be amplicon contamination from the lab if you have recently processed amplicons in those sizes recently in the same location.

evobio721 12-22-2017 08:40 AM

Quote:

Originally Posted by nucacidhunter (Post 213574)
It does not seem to be result of over-amplification. Most likely cassette lane has been faulty or other weak cause could be amplicon contamination from the lab if you have recently processed amplicons in those sizes recently in the same location.

That sounds reasonable to me. Thank you very much for your time and help. I greatly appreciate it.


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