Hello,
I am a bit confused on how to calculate the volume of PhiX to add to the sample prior to sequencing on the Illumina platform. Based on the Illumina protocols for the MiSeq for example, If I am loading a total volume of 600 ul of which 300 ul of denatured pool and 300 ul of HT1, am I supposed to calculate the volume of PhiX on the total volume of 600 ul or based on the volume of sample I am using, in this case 300 ul? Based on the protocols, Illumina says to add 6 ul of PhiX in a 600 ul of final solution to achieve a PhiX spike of 1%. However, when I did this, on the SAV files, the % of aligned PhiX is always around the double of the amount. I am a bit confused because I am dealing with incredibly low diversity libraries that will yield to low reads passing filters, and would like to avoid adding a 30% PhiX (thinking of adding only 15%) because the PhiX will take up even more reads reducing again the amount of sample reads. I would really appreciate if anyone could help me understand.
Thanks and happy sequencing to all of you!
I am a bit confused on how to calculate the volume of PhiX to add to the sample prior to sequencing on the Illumina platform. Based on the Illumina protocols for the MiSeq for example, If I am loading a total volume of 600 ul of which 300 ul of denatured pool and 300 ul of HT1, am I supposed to calculate the volume of PhiX on the total volume of 600 ul or based on the volume of sample I am using, in this case 300 ul? Based on the protocols, Illumina says to add 6 ul of PhiX in a 600 ul of final solution to achieve a PhiX spike of 1%. However, when I did this, on the SAV files, the % of aligned PhiX is always around the double of the amount. I am a bit confused because I am dealing with incredibly low diversity libraries that will yield to low reads passing filters, and would like to avoid adding a 30% PhiX (thinking of adding only 15%) because the PhiX will take up even more reads reducing again the amount of sample reads. I would really appreciate if anyone could help me understand.
Thanks and happy sequencing to all of you!
Comment