Alternating High/Low intensity of sample, but not PhiX

BBthekid007 · 02-21-2012, 11:51 AM

I have a pretty strange problem that I haven't seen discussed here before. I made an amplicon library using the multiplexed primers described on these forums and have begun a single-lane HiSeq run. The resulting amplicons are about 260 bases in length. The run is still in progress (PE-100), but the results from the first read are very weird. The first position of the first read has virtually no sample intensity, although the spiked PhiX shows up nicely. This continues for a position or two, followed by very high intensity from the sample (and the PhiX). The alternating sample intensity continues throughout the first read, but the spiked PhiX stays consistent. The other lanes on the run seem to be fine. What's wrong with my sample?

PS: I'm not doing the sequencing myself (the university core lab is doing it).

-Bryan

Rocketknight · 02-22-2012, 03:55 AM

When you say very high intensity from the sample and the PhiX, does that mean the PhiX intensity also goes up on that cycle? Or just that it remains at the same high level it was at initially?

HESmith · 02-22-2012, 06:36 AM

Do you mean the intensities of all four bases fluctuate by cycle, or are you looking at only a single base (I think A is shown by default)?

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02-21-2012, 11:51 AM	#1
BBthekid007 Junior Member Location: Nashville Join Date: Feb 2010 Posts: 4	Alternating High/Low intensity of sample, but not PhiX I have a pretty strange problem that I haven't seen discussed here before. I made an amplicon library using the multiplexed primers described on these forums and have begun a single-lane HiSeq run. The resulting amplicons are about 260 bases in length. The run is still in progress (PE-100), but the results from the first read are very weird. The first position of the first read has virtually no sample intensity, although the spiked PhiX shows up nicely. This continues for a position or two, followed by very high intensity from the sample (and the PhiX). The alternating sample intensity continues throughout the first read, but the spiked PhiX stays consistent. The other lanes on the run seem to be fine. What's wrong with my sample? PS: I'm not doing the sequencing myself (the university core lab is doing it). -Bryan Last edited by BBthekid007; 02-21-2012 at 01:04 PM.

02-22-2012, 03:55 AM	#2
Rocketknight Member Location: Ireland Join Date: Sep 2011 Posts: 78	When you say very high intensity from the sample and the PhiX, does that mean the PhiX intensity also goes up on that cycle? Or just that it remains at the same high level it was at initially?

02-22-2012, 06:36 AM	#3
HESmith Senior Member Location: Washington DC Join Date: Oct 2009 Posts: 280	Do you mean the intensities of all four bases fluctuate by cycle, or are you looking at only a single base (I think A is shown by default)?