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Ion Chip™ Minifuge.
Tank of compressed nitrogen.
Glass bottle (1 L).
NaOH (10 M).
96-Well Thermal Cycler.
Fluorometer (Qubit™).
Eppendorf™ DNA LoBind™ Microcentrifuge Tubes, 1.5 mL.
Agencourt™ AMPure™ XP Kit.
DynaMag™-96 Side Magnet or other plate magnet.
Nuclease-free Water.
Ethanol.
Pipettors, 2–200 μL, and low-retention filtered pipette tips.3 Methods
Overview of Next-generation sequencing: Next-generation
sequencing involves a wet and dry laboratory workflow for its
application in a research or diagnostic setting. We describe the
workflow of targeted sequencing using a benchtop sequencer. A
number of different library preparation kits are commercially avail-
able. Each one makes use of similar molecular techniques and only
varies slightly. The quality of DNA greatly influences the efficacy of
library preparation and ultimately sequence output.
Before Library Preparation:
●● Minimize freeze-thaw cycles of panels by aliquoting as
needed for your experiments.
●● Panels can be stored at 4 °C for 1 year.
●● Use good laboratory practices to minimize cross-contam-
ination of products.
●● Always change pipette tips between samples.
●● Pipet viscous solutions slowly and ensure complete mixing
by vortexing or pipetting.
●● Thaw components that contain enzymes on ice, and keep
them on ice during procedure.
●● All other components, including primer pools, can be
thawed at room temperature.
●● Gently vortex and centrifuge before use.- Isolate genomic DNA or RNA.
- If starting with RNA, reverse-transcribe to make cDNA.
- For panels with two primer pools, use the following table to
prepare for each sample a target amplification master mix with-
out primers in a 1.5-mL tube (see Note 1).
3.1 Construction
of Library
Suja Pillai et al.