Hello,
I have been running a Miseq instrument for just under a year now, and received the upgrade in October. I run a variety of amplicon and whole genome samples prepared by various collaborators of mine. I always spike 25-40% PhiX into the amplicon samples, as they tend to be low diversity. I never gave the "% aligned" metric much thought before the upgrade because it always accurately reflected the amount of PhiX added (i.e. if I added 30% phiX to my amplicon pool, the % aligned to the PhiX genome was always ~30%).
Recently, the "% aligned" values on my amplicon runs have ranged from 1% to 98% despite the fact I have been adding 25-40%. I have not been able to correlate anything sample-related to this phenomenon (DNA concentration, sample source, amplicon type, lab preparing the samples, cluster density, reads PF, etc...) and am now seeking to understand this "% aligned" metric a bit better. Most concerning to me is that I recently ran a 100% PhiX run on the instrument and only 63% of the reads aligned to the PhiX genome. What could cause this?
I am new to this forum and attempted to search for answers to this issue as best I could, but if you know of an existing thread related to this matter, please let me know!
Any insight is greatly appreciated!
I have been running a Miseq instrument for just under a year now, and received the upgrade in October. I run a variety of amplicon and whole genome samples prepared by various collaborators of mine. I always spike 25-40% PhiX into the amplicon samples, as they tend to be low diversity. I never gave the "% aligned" metric much thought before the upgrade because it always accurately reflected the amount of PhiX added (i.e. if I added 30% phiX to my amplicon pool, the % aligned to the PhiX genome was always ~30%).
Recently, the "% aligned" values on my amplicon runs have ranged from 1% to 98% despite the fact I have been adding 25-40%. I have not been able to correlate anything sample-related to this phenomenon (DNA concentration, sample source, amplicon type, lab preparing the samples, cluster density, reads PF, etc...) and am now seeking to understand this "% aligned" metric a bit better. Most concerning to me is that I recently ran a 100% PhiX run on the instrument and only 63% of the reads aligned to the PhiX genome. What could cause this?
I am new to this forum and attempted to search for answers to this issue as best I could, but if you know of an existing thread related to this matter, please let me know!
Any insight is greatly appreciated!
Comment