Agilent eBook - 22

NGS Library QC Steps
to Ensure Successful

easily accomplished using automated electrophoresis
instruments. Once these values are determined, the
molarity can be calculated, allowing for equal molar
pooling and proper flow cell loading. Due to these
factors, all major sequencing instrument providers
and library preparation kit manufacturers recommend
completing QC steps on the starting raw materials,
throughout the library preparation process, all the way
to the final library.

GEN interview with Kyle Luttgeharm, PhD, Product
Manager, Parallel CE Instruments, Diagnostics and
Genomics Group, Biomolecular Analysis Division, Agilent

GEN: I hear there are quality metrics that can
help identify samples. What are they and how
are they used?

GEN: Why is NGS Library QC so important for
successful sequencing?
KL: Quality control ensures a complete understanding
of the starting material, the success of library preparation steps, and the quality of the final library. By having
robust quality control procedures, you can be confident
in your library preparation prior to flow cell loading.
A quality sample is based upon the sample type
and application. DNA perfectly suited for short-read
sequencing applications may be considered of very
low quality for long-read applications. Additionally, a
high quality FFPE sample would be considered a highly
degraded sample from most other sources.
Electrophoretic analysis of samples lets you decide
if the starting material is suitable for individualized
applications. When you start a sequencing project it is
important to understand the quality of the raw material



Kyle Luttgeharm, PhD

as this impacts raw material processes such as shearing. By completing quality control steps throughout
the library preparation process proper removal of
impurities such as adapter dimers and primer dimers
is ensured ultimately allowing for the creation of a
symmetrical, artifact-free library for sequencing.
When sequencing library pools it is important that
you have equal molar concentrations of each library
for proper flow cell loading. This ensures that all
your libraries are equally represented. To calculate
the molarity, you must determine both the average
size and concentration of your samples. This can be

KL: Quality metrics provide an objective look at your
data. They remove the individual scientist's biases
regarding whether or not it is a quality sample. Raw
RNA material is where quality metrics are most widely
used. Agilent has developed quality metrics for all of
our automated electrophoresis instruments, including
the RNA Integrity Number (RIN) for the Bioanalyzer
system, the RNA Integrity Number Equivalent (RINe)
for the TapeStation systems, and RNA Quality Number
(RQN) for the Fragment Analyzer and Femto Pulse
systems. Each provides a sample a score based on
a scale of 1-10, with 10 being very intact RNA and 1
being highly degraded RNA. Due to the difference in the
technology used, each system uses a unique algorithm
to calculate the RNA quality score; however, they all

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