Monday 10/19/15

Set-up a MiSeq run with the 2b-rad library (concentration of .815 ng/uL) finished on Friday 10/16/15. I’m using the MiSeq in the Pritzker DNA Lab at the Field Museum and a v3 reagent kit. To prepare the library for sequencing, I followed Illumina’s instructions. These instructions 1st call for a 4nM library, which I prepared based off these instructions: CALCULATIONSANDCONVERSIONSINPREPARATIONFORIlluminaMiseqRUN. The fragments should be approximately 176bp, so I used the following formula to get the nM conversion factor: 10^6/660/N (where N is the size of the fragment in basepairs). Taken from this website. This formula gave 8.6 which I rounded to 8. Therefore, to dilute my library:

  • 8nM*.815ng/uL = 6.5nM
  • Dilution: 6.5nM/4nM = 1.625 -> 1 uL sample + .625 water
  • Dilution x 5: 5 uL sample + 3.125 water = 8.125 total volume.

While preparing the library, Illumina gives options for the PhiX spike-in and the concentration to run on the flow cell. I chose a 2% PhiX spike in and 10pM as previous users of this MiSeq have reported overclustering at 11pM.

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