Improved Nucleic Acid QC with Parallel CE - 8

Application Note

Best Sizing Practices

Quality Metrics

Summary
The Fragment Analyzer provides accurate and consistent sizing for DNA fragments and smears. Peak size, ideal for fragments, is
automatically provided by PROSize, while the PROSize Smear Analysis Tab provides an average size for smears based on the distribution
of the sample over the designated smear range. Sizing under 6,000 bp is accurate and consistent across the concentration range of the
respective kits, with reliable sizing over 6,000 bp occurring with the recommended sample concentrations. High salt concentrations
in samples minimally affect sizing, still allowing for exceptional precision and accuracy.

Introduction
Advanced Analytical Technologies, Inc. (AATI) uses four quality metrics, the DQN, GQN, RQN, and DV200 to assess the quality and
integrity of DNA and RNA. These metrics have been published in numerous articles and incorporated in protocols and literature by
Illumina®, PacBio®, and Bionano Genomics®.
DQN and GQN

Kits Used in Study:
* DNF-477 High Sensitivity Small Fragment Analysis Kit
* DNF-474 High Sensitivity NGS Fragment Analysis Kit
* DNF-464 High Sensitivity Large Fragment 50Kb Analysis Kit

AATI designed the DNA Quality Number (DQN) and the Genomic Quality Number (GQN) for use in PROSize® Data Analysis Software
to allow for easy analysis of sheared DNA and genomic DNA (gDNA) quality, respectively. The user defines a Size Threshold they deem
appropriate for their specific application. PROSize then calculates a DQN or GQN value based upon the fraction of the total measured
concentration of the sample that lies above the specified Size Threshold. The DQN and GQN scores samples on a scale of 0 to 10, with
0 indicating none of the sample exceeds the threshold and 10 indicating 100% of the sample lies above the threshold value. Since DNA
size can vary due to numerous factors, such as: origin of organism, extraction methods, tissue type, and sample handling; the ability
to define the Size Threshold gives the user the advantage of defining quality DNA for their specific application.

Written by: Chava Pocernich, Ph.D., Ben Deist M.S.; Whitney Pike, M.S.; Denise Warzak, M.S.
Email: techdata@aati-us.com

GQN is used specifically for genomic DNA samples separated on the Fragment Analyzer™ Automated CE System with the DNF-467
Genomic DNA 50 Kb Analysis Kit and DNF-488 High Sensitivity Genomic DNA Analysis Kit and on the FEMTO Pulse® Automated
Pulsed-Field CE Instrument with the FP-1002 gDNA 165 Kb Analysis Kit (gDNA 165 Kb Kit)1. The DQN is used for all other DNA sample
types and was created for samples that use a reagent kit with both a lower and upper marker.

For additional application notes see our website: www.aati-us.com/application-notes

As seen in Figure 1, sheared gDNA samples from PacBio® of varying sizes were separated on the FEMTO Pulse with the gDNA 165 Kb
Kit. The Size Threshold for these samples was set at 30,000 bp and reported a lower GQN for the smaller sized samples compared to
the larger sized gDNA, as expected due to the varying size. The DQN and GQN values allow for fast and easy assessment of the quality
of any type of DNA sample.

Fragment Analyzer™ Automated CE System
The Fragment Analyzer by Advanced Analytical Technologies, Inc. offers
researchers the ability to qualify and quantify nucleic acid samples quickly
and reliably through parallel capillary electrophoresis. It was designed to
solve laboratory bottlenecks and quicken the time to results by:
*
*
*

B
LM

Variable Throughput - scalable array with 12, 48,
or 96 capillaries
High Separation Resolution - detection limits as
low as 5 pg/µL for DNA fragments
High Sensitivity - achieve as low as 3 bp resolution

Ave. Smear Size
(bp)

GQN
Set at 30Kb

12,147

23,339

1.5

45,304

6.4

57,789

7.1

73,267

7.8

94,045

7.8

109,968

8.2

164,292

8.8

Figure 1: Sheared gDNA from PacBio® separated on the FEMTO Pulse with the FP-1002 gDNA 165 Kb Analysis Kit. (A) Separations on the
electropherogram (B) Average smear size, and GQN30,000 , n=2, LM = Lower Marker.
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As a dynamic quality metric, the GQN brings objective analysis to the quality assessment of formalin-fixed, paraffin-embedded
(FFPE) gDNA samples. FFPE DNA undergoes substantial chemical modifications during formalin fixation, including crosslinking and
fragmentation. Utilizing AATI's Fragment Analyzer, Illumina developed three guidelines to follow based on the GQN value when
preparing an FFPE library: (1) minimal DNA input into library preparation, (2) number of PCR cycles, and (3) the amount of library used
in enrichment2. Successful and reliable library preparations and sequencing results can be achieved with the GQN metrics.

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Copyright ©2018 Advanced Analytical Technologies, Inc. All Rights Reserved. Reproduction, adaptation or translation without prior written permission is prohibited except as allowed under
copyright laws. Fragment Analyzer™ is a trademark of Advanced Analytical Technologies, Inc. Prosize® is a registered trademark of Advanced Analytical Technologies, Inc. All products and
services described herein are intended FOR RESEARCH ONLY and NOT FOR USE IN DIAGNOSTIC PROCEDURES.

www.aati-us.com

1 https://www.aati-us.com/femto-pulse-gdna-qc/ FEMTO Pulse® Genomic DNA Sizing and Quality Control
2 https://www.illumina.com/content/dam/illumina-marketing/documents/products/technotes/truseq-exome-ffpe-tech-note-770-2015-035.pdf

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Improved Nucleic Acid QC with Parallel CE

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