eBook: Optimizing Results from Nucleic Acid Isolation - 11

with the accelerating adoption of sequencing in clinical applications, such as cancer diagnostics where
early and reliable diagnosis can impact long-term
patient outcomes.

human tissue samples. These FFPE samples, for example, might require an extra step to repair damaged DNA to make them suitable for sequencing.
Sample preparation can also include an amplification step to increase the amount of nucleic acid fragments to a level sufficient for library preparation and
sequencing instruments.

Genomic sequencing companies now offer both
public and private NGS services for genetic testing.
To remain cost-efficient and cost-effective, these
companies need to maintain a high sample throughput, processing large numbers of NGS samples per
day and keeping their sequencing systems busy
churning out data.

Library preparation takes the purified and amplified
nucleic acid (DNA or RNA) from sample prep and
processes them to be of optimum length by adding
sequencing barcodes and adaptors. In some cases, a
hybridization- or PCR-based enrichment step further
optimizes the library for sequencing by enriching
the specific regions of interest.

The difference between NGS sample
prep and library prep

The different approaches to NGS
sample preparation

Between sample collection and sequencing in an
NGS workflow, there are two key phases: sample
preparation and library preparation, each containing a series of steps (Figure 1).

There are various technologies and approaches
enabling sample preparation for NGS. These range
from traditional solution-based protocols, through
solid-phase and membrane chemistries, to magnetic bead-based approaches.

NGS sample preparation involves sample-specific
steps for extracting nucleic acids, such as genomic
DNA from formalin-fixed, paraffin-embedded (FFPE)

Magnetic bead-based approaches are arguably a
subcategory of solid-phase extraction, but there
are numerous chemistries possible, and the workflow is distinct enough to consider as a separate
type altogether.
Each of these approaches follows the three basic
steps to DNA (or RNA) isolation:
* Lysis of cells in sample, varying from gentle
detergents to aggressive homogenization.
* Removal of contaminants, including unwanted
nucleic acids and denatured proteins.

Figure 1: Typical NGS workflow from sample collection to
sequencing and analysis. Sample preparation (mid-blue)
approach can vary considerably, dependent on the source,
quantity, and quality of starting material.

* Recovery of nucleic acids into an appropriate
buffer for any downstream application.

11


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eBook: Optimizing Results from Nucleic Acid Isolation

Table of Contents for the Digital Edition of eBook: Optimizing Results from Nucleic Acid Isolation

Contents
eBook: Optimizing Results from Nucleic Acid Isolation - 1
eBook: Optimizing Results from Nucleic Acid Isolation - Contents
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