Hi all,
I have come across issues with the Illumina PhiX v3 in my project. I need to sequence my low diverse library on NextSeq 500. To balance out the diversity, I tried to spike-in 50% of the Illumina PhiX v3 control along with my library. But, the run wasn't good and I only managed to see a 9% alignment in my first run. At first, I thought it was resulted from the degradation of my PhiX. Thereby, I quantified my PhiX using picrogreen and the reading seems fine after conversion and the bioanalyzer showed no sign of degradation. I repeated my run the 2nd time. This time the Illumina FAS came over and supervised my library denaturation method to ensure I followed exactly the instructions recommended in the protocol, ie. library denaturation->library dilution to 1.8pM followed by PhiX denaturation->dilution of denatured PhiX to 1.8pm and, eventually, mixed both library and PhiX at 50:50 ratio. Everything was fine but the results of the 2nd run was still the same as in we couldn't achieved near 50% of PhiX spike-in. In addition, we observed a high error rate too. Does anyone have any suggestion on this issue? Thank you in advance for the guidance.
I have come across issues with the Illumina PhiX v3 in my project. I need to sequence my low diverse library on NextSeq 500. To balance out the diversity, I tried to spike-in 50% of the Illumina PhiX v3 control along with my library. But, the run wasn't good and I only managed to see a 9% alignment in my first run. At first, I thought it was resulted from the degradation of my PhiX. Thereby, I quantified my PhiX using picrogreen and the reading seems fine after conversion and the bioanalyzer showed no sign of degradation. I repeated my run the 2nd time. This time the Illumina FAS came over and supervised my library denaturation method to ensure I followed exactly the instructions recommended in the protocol, ie. library denaturation->library dilution to 1.8pM followed by PhiX denaturation->dilution of denatured PhiX to 1.8pm and, eventually, mixed both library and PhiX at 50:50 ratio. Everything was fine but the results of the 2nd run was still the same as in we couldn't achieved near 50% of PhiX spike-in. In addition, we observed a high error rate too. Does anyone have any suggestion on this issue? Thank you in advance for the guidance.
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