Hello!
I have performed a phiX validation run with Illumina standard phiX kit and V3 chemistry. I diluted the phiX library to 10 pM and expected to get a cluster density of around 1000, but it resulted in a very low amount of cluster density (~130). Also as you can see in SAV plots, phred scores have decreased after cycle 100 in read 1 and cycle 40 in read 2, which resulted in a low >Q30 percentage at the end of the run. What do you think has caused this issue and how I can fix it? Can this be because of the low cluster density? can this be because of bad reagent storage conditions or handling? I have performed a system check and it was successful. what are your suggestions?
I have attached plots of SAV analysis and thumbnail images in different cycles of the run. the photo with better quality is for cycle #17 and the photo with lower quality is for cycle #436, both for A nucleotide.
Thank you.
It's hard to tell, but it looks as if your kit has expired. You should look at phasing and prephasing values, if they are high it indicates a chemistry problem.
I would recommend to contact Illumina customer support related to this problem.
Marcin Golebiewski thanks, the legacy phasing/prephasing rate is 0.430/0.105 for read1 and 0.507/0.155 for read 2. in comparison to my other runs I assume this is high. Am I right?
Yes, this is high, but not very high. Thus, your kit was bad, but not very bad ;) This is higher than the recommended values of < 0.1% in read1, but it is not that bad, as I've seen values of 1% in some runs. If your kit was within the expiry period, I'd try to get a replacement one from Illumina.
Marcin Golebiewski Thanks a lot for your help, what must be the recommended value for read2?
As far as I know, there is no official recommended value, but one should expect both phasing and prephasing in read2 to be slightly greater than in read1.
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