PacBio Onso - Page 14

Page 14
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PN 103-140-400 REV05 JUN2024

Step
3b. Denature and dilute DNA library WITH LQC control library spike-in
3b.1
Retrieve a 1 mL tube of DIL buffer from 4°C storage.
3b.2
Calculate the LQC concentration required per lane as follows in the table below.
For libraries with low sample diversity, it is advised to increase the percent spike-in of LQC up to 20%,
whereas otherwise a standard 5% spike-in amount is suitable.
Refer to the section “Library loading concentration and control spike-in guidelines” under “General best
practices” for sample library and LQC loading concentration recommendations.
Calculating LQC spike-in concentration (per lane)
c = a * (b/100)
User input
a
Desired total library loading concentration (pM)
b
LQC spike-in percent (%)
c
LQC spike-in concentration (pM)
3b.3
Calculate the LQC input volume required per lane as follows:
Calculating LQC input volume (per lane)
f = (c * e)/d
User input
c
LQC spike-in concentration (pM)
d
LQC starting concentration (pM)
2000
e
Final library volume (µL)
380
f
Volume of starting LQC library (µL)
It is recommended to have an LQC input concentration of at least 500 pM, but this value can
be increased if the volume of LQC + DNA library > 16 µL.
3b.4
Calculate the sample library concentration required per lane as follows:
Calculating sample library concentration (per lane)
g = a - c
User input
a
Desired total loading concentration (pM)
c
LQC spike-in concentration (pM)
g
Final sample library concentration (pM)