PacBio Onso - Sequencing Procedure; Thawing Sequencing Reagent Pack Thaw; Optional Custom Primer Addition

Page 31
© 2024 PacBio. All rights reserved. For research use only. Not for use in diagnostic procedures.
PN 103-140-400 REV05 JUN2024
Sequencing procedure
1. Thawing sequencing reagent pack thaw
An Onso sequencing reagent pack can be removed from the box and mylar bag prior to thaw, as described below.

Step
Instructions
1.1
Remove the sequencing reagent pack from -20°C storage. Remove the reagent pack from packaging and the
mylar bag. Discard the packaging and mylar bag.
1.2
Check that the foil seals of each reagent bottle are intact by visual inspection of the top of the reagent pack. If
not, dispose of the reagent pack in accordance with your site chemical waste disposal practices and obtain a
new reagent pack.
1.3
Thaw the sequencing reagent pack in one of two ways:
• Thaw at room temperature (18-25°C) for 16 to 24 hours prior to sequencing.
• Thaw the reagent pack in a room temperature (18-25°C) water bath with circulating water set to 25°C for 2
hours and remove promptly.
Water bath depth should be 4-6 inches when the reagent pack is in the water bath. Ensure
thawing in the water bath does not exceed more than 2 hours.
1.4
Dry the thawed reagent pack with a towel before proceeding. Be sure to wipe any condensation or water off
the bottles in addition to the exterior of the reagent pack plastic clamshell.
1.5
Firmly grip the ends of the reagent pack with both hands. Thoroughly mix the contents by inverting the
sequencing reagent pack at least 10 times. Visually inspect to confirm the reagent pack is fully thawed and
mixed.
2. Optional custom primer addition
Custom primers should be added to the thawed reagent pack when sequencing converted libraries. If not using
custom primers for sequencing, then proceed to section 3 to set up sequencing. Tube numbers indicated in
magenta refer to the reagent pack position in the table below.
2a. Custom primer addition for sequencing pre-existing P5/P7 libraries clustered with Onso indexed
library control spike-in
Description Primer
Custom Primer to add
Reagent Pack Position
Insert Read 1 Primer
Custom Insert Read 1 Primer
8
Index 1 Primer (downstream of the insert)
Custom Index Read 1 Primer
7
Insert Read 2 Primer
Custom Insert Read 2 Primer
6
Index 2 Primer (upstream of the insert)
Custom Index Read 2 Primer
5