Improved Nucleic Acid QC with Parallel CE - 4

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Importance of QC for the reliable preparation of NGS libraries
Advanced Analytical Technologies, Inc. 2450 SE Oak Tree Ct. Ankeny, IA 50021 USA

Introduction
Nucleic acid QC is an essential component of next-generation sequencing workflows. Confirming nucleic acid quality at checkpoints
throughout NGS library preparation - from incoming nucleic acid QC to final library validation - helps ensure successful sequencing
outcomes.
Advanced Analytical Technologies, Inc. (AATI) addresses the QC needs of researchers performing NGS with a combination of parallel
capillary electrophoresis and fluorescence detection of nucleic acids. The Fragment Analyzer™ Automated CE System uses thoughtful
design considerations to provide superior nucleic acid detection and separation capacity, while maintaining ease-of-use. Easily size,
quantify, and qualify DNA and RNA samples without swapping capillary arrays, and complete analytic separations in about one hour
with the gold standard of nucleic acid QC.

stem from poor separation quality, limiting accurate sizing and quantification of important regions, including the Fast Region, small rRNA
subunits, and large rRNA subunits. Superior separation parameters and exceptional sensitivity delivered by the Fragment Analyzer
provide researchers with a complete picture of total RNA quality, helping ensure only suitable samples proceed to downstream analysis.

Incoming Nucleic Acid QC
NGS commonly begins with isolation of genomic DNA or total RNA. Knowing the quality of nucleic acid samples prior to NGS library
construction is critical. Knowing the quality of DNA and RNA helps ensure optimal NGS library preparation, in turn supporting successful
sequencing. Accurate QC of DNA and RNA samples requires an accurate understanding of the size and concentration, as well as visual
confirmation of sample quality.

Post-separation analysis of total RNA is greatly simplified by PROSize. Using the RNA Quality Number (RQN), researchers are able to
quickly, and objectively, determine which total RNA samples are suitable for NGS library preparation. Automatically provided by PROSize
in the RNA Property Summary, the RQN is reported on a scale of one to 10, with higher values corresponding to higher quality total RNA.
While the RQN delivers accurate objective analysis of total RNA expected to be of good quality, it has limited application with FFPE RNA
samples often studied in clinical research settings. To this end, the DV200 quality metric was developed1. Available for PROSize, the
DV200 reports the percentage of RNA fragments that exceed 200 nt in size. Notably, this quality metric has been found to have a direct
correlation with a samples relative performance in NGS workflows. The DV200 helps researchers obtain critical results from FFPE RNA
sequencing projects by ensuring only suitable samples proceed to NGS.

Genomic DNA
Genomic DNA is a common starting point for numerous NGS applications. QC analysis of gDNA is frequently complicated by the high
molecular weight of the sample, impeding sizing and quantification. By employing a combination of specially designed separation
matrices and carefully programmed separation methods, the Fragment Analyzer line of instruments can reliably separate gDNA (Figure
1) with accurate sizing through 60 Kb in about one hour.

Final Library Validation

Objective QC analysis of gDNA samples post-electrophoresis is aided by the Genomic Quality Number (GQN), a dynamic quality metric
available in PROSize® Data Analysis Software. Calculated from a user-defined Size Threshold, the GQN reflects the quantity of gDNA
that meets unique, user-defined standards. Reported on a scale of 0 to 10, a higher value indicates that more of the gDNA sample falls
above the Size Threshold.

Final QC analysis of NGS libraries prior to sequencing ensures that library construction was successful. It is also when researchers will
calculate the molarity of the library from its measured average size and concentration. NGS flow cells require precision loading to help
guarantee the generation of high-quality sequencing results. The Fragment Analyzer can simultaneously provide visual confirmation of
library quality, and calculate molarity for short-read and long-read NGS libraries.

FFPE gDNA is a valuable resource, though it presents unique QC challenges. The GQN can quickly evaluate the suitability of FFPE gDNA
samples separated on the Fragment Analyzer. Degraded by tissue preservation, FFPE gDNA samples evade accurate analysis. The
Fragment Analyzer provides complete separation of these samples, while the GQN allows researchers to define what constitutes suitable
size for FFPE samples moving on to NGS.

Short-read NGS (Figure 3) is perhaps one of the most common sequencing strategies in use today. While chemistries differ, all shortread NGS relies on the use of libraries comprised of relatively short DNA fragments. Frequently employed in high-throughput workflows,
short-read NGS demands a QC solution that can adapt to different throughput demands without sacrificing accuracy or precision. The
Fragment Analyzer automates QC analysis, processing 12, 48, or 96 samples in an hour and up to 288 samples without researcher
intervention.

Total RNA

In contrast to short-read NGS, long-read NGS is characterized by employing large-insert libraries. These libraries can use DNA inserts
tens of kilobases long (Figure 4), simplifying post-sequencing assembly and analysis. While long-read sequencing has its benefits, it also
has QC challenges. Primarily, the size of the library defies accurate sizing and quantification by legacy methods. The Fragment Analyzer
can size and quantify DNA smears, including long-read NGS libraries up to 48.5 Kb, in about 1 hour, considerably faster and with higher
accuracy and precision than competing instruments.

Total RNA (Figure 2) contains a wealth of information ready for mining with NGS strategies. QC analysis of total RNA is crucial, as use
of degraded or otherwise low-quality samples for NGS library construction can compromise success. Challenges with total RNA QC

Summary
The Fragment Analyzer is the gold standard of nucleic acid QC for NGS workflows, simplifying necessary QC analysis from incoming
nucleic acids to final library validation. DNA and RNA samples are easily separated using the same capillary array, while automated
instrument operation and parallel capillary electrophoresis bring researchers an efficient, accurate, and easy-to-use QC solution.

References
Evaluating RNA Quality from FFPE Samples, Illumina®.

https://www.illumina.com/content/dam/illumina-marketing/documents/products/technotes/evaluating-rna-quality-from-ffpe-samplestechnical-note-470-2014-001.pdf
BioTechniques 64:-81-82 (February 2018) doi 10.2144/000114495

Vol. 64 | No. 2 | 2018

www.BioTechniques.com

Vol. 64 | No. 2 | 2018

www.BioTechniques.com

https://www.illumina.com/content/dam/illumina-marketing/documents/products/technotes/evaluating-rna-quality-from-ffpe-samples-technical-note-470-2014-001.pdf https://www.illumina.com/content/dam/illumina-marketing/documents/products/technotes/evaluating-rna-quality-from-ffpe-samples-technical-note-470-2014-001.pdf

Improved Nucleic Acid QC with Parallel CE

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