Extra "C" Peaks

kbusseq · 02-17-2015, 10:47 AM

The last plate I ran on our ABI 3730 had a very strange problem with the traces that I haven't seen before and I can't seem to find anything about it online either. Every time there was a "C" base call, the following peak had a smaller "C" peak underneath it. It seemed to be template-independent as I had DNA from a number of different projects (different extraction methods, different people doing the extractions, etc.) on the same plate and they all had this problem. The plates we'd run just the day before looked great - and even these plates weren't horrible, they just had lower quality than normal because of these strange extra "C" peaks.

Any idea what might be causing this? I've read that BigDye issues tend to affect "G" bases first, array issues (in my experience anyway) tend to affect the timing and spacing of the peaks, and salt contamination just lowers signal strength in general, so none of those are likely to be the problem, and I can't think of anything else that would be template-independent.

Thanks in advance!

microgirl123 · 02-18-2015, 07:26 AM

It does sound like a degradation issue with your BigDye. ABI has a really useful guide to all sorts of problems:
https://www3.appliedbiosystems.com/c...cms_041003.pdf

It could be that the degradation is not in the BigDye stock, but is happening after you prep your samples. We always cover ours with mineral oil to prevent problems like this.

kbusseq · 02-18-2015, 09:21 AM

Thank you so much! I'd been trying to find more info through ABI's documents but hadn't come across that one - it'll be a great resource to have in the future.

nextgen · 10-08-2015, 08:57 AM

Have you solved your extra "c" probkem? It is C-shoulder problem caused by purification process. If you use aline biosciences Pureseq, the problem would be eliminated.

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02-17-2015, 10:47 AM	#1
kbusseq Junior Member Location: Tempe, AZ Join Date: Mar 2013 Posts: 7	Extra "C" Peaks The last plate I ran on our ABI 3730 had a very strange problem with the traces that I haven't seen before and I can't seem to find anything about it online either. Every time there was a "C" base call, the following peak had a smaller "C" peak underneath it. It seemed to be template-independent as I had DNA from a number of different projects (different extraction methods, different people doing the extractions, etc.) on the same plate and they all had this problem. The plates we'd run just the day before looked great - and even these plates weren't horrible, they just had lower quality than normal because of these strange extra "C" peaks. Any idea what might be causing this? I've read that BigDye issues tend to affect "G" bases first, array issues (in my experience anyway) tend to affect the timing and spacing of the peaks, and salt contamination just lowers signal strength in general, so none of those are likely to be the problem, and I can't think of anything else that would be template-independent. Thanks in advance!

02-18-2015, 07:26 AM	#2
microgirl123 Senior Member Location: New England Join Date: Jun 2012 Posts: 200	It does sound like a degradation issue with your BigDye. ABI has a really useful guide to all sorts of problems: https://www3.appliedbiosystems.com/c...cms_041003.pdf It could be that the degradation is not in the BigDye stock, but is happening after you prep your samples. We always cover ours with mineral oil to prevent problems like this.

02-18-2015, 09:21 AM	#3
kbusseq Junior Member Location: Tempe, AZ Join Date: Mar 2013 Posts: 7	Thank you so much! I'd been trying to find more info through ABI's documents but hadn't come across that one - it'll be a great resource to have in the future.

10-08-2015, 08:57 AM	#4
nextgen Member Location: East Coast Join Date: Feb 2010 Posts: 26	Have you solved your extra "c" probkem? It is C-shoulder problem caused by purification process. If you use aline biosciences Pureseq, the problem would be eliminated.