Templated bead preparation was performed according to the “full scale” protocol.
Bead counts are calculated as follows:
Avg bead count x # hemacytometer squares x volume in hemacytometer (uL) x dilution factor = beads/uL x suspension volume (uL) = total beads
Initial Bead counts: (1:200 dilution)
CT: 132, 133, 127, 136 Avg. = 132
WB: 127, 128, 119, 126 Avg. = 125
Lean: 121, 114, 132, 109 Avg. = 119
CT: 132 x 25 x 10 x 200 = 6.6x10^6 beads/uL x 200uL = 1.32x10^9 beads
WB: 125 x 25 x 10 x 200 = 6.25x10^6 beads/uL x 200uL = 1.25x10^9 beads
Lean: 119 x 25 x 10 x 200 = 5.95x10^6 beads/uL x 200uL = 1.19x10^9 beads
Templated Bead counts (1:10 dilution)
CT: 91, 80, 100, 78 Avg. = 87.25
WB: 69, 70, 75, 65 Avg. = 69.75
Lean: 40, 52, 48, 46 Avg. = 46.5
CT: 87.25 x 25 x 10 x 10 = 218125 beads/uL x 400uL = 8.7525x10^7 beads
WB: 39.75 x 25 x 10 x 10 = 174375 beads/uL x 400uL = 6.975x10^7 beads
Lean: 46.5 x 25 x 10 x 10 = 116250 beads/uL x 400uL = 4.65x10^7 beads
Percent Recovery Templated Beads
CT: (8.7252x10^7 beads)/(1.32x10^9 beads) x 100 = 6.61%
WB: (6.975x10^7 beads)/(1.25x10^9 beads) x 100 = 5.58%
Lean: (4.65x10^7 beads)/(1.19x10^9 beads) x 100 = 3.91%
Results: Everything looks pretty darn good. One mild concern, however, is the yield from the the Lean library. An 8-well slide requires 41 million beads for a run. Additionally, I believe 15 million are needed for a WFA (quality check, pre-run). This means that the Lean prep is nearly 10 million beads short of what is necessary for a “complete” run of this sample. Will send the numbers to Rhonda and see what her opinion is and what she suggests to do. But, based on the percent recovery, all the samples should be really high quality (extremely few polyclonal beads).