Improved Nucleic Acid QC with Parallel CE - 6

Application Note

Best Sizing Practices

Best Sizing Practices with the Fragment Analyzer™

similar peak and smear sizes (Figure 1A). In contrast, DNA smears with a larger distribution after the highest peak reported a larger
smear size compared to the peak size as expected (Figure 1B). In the same respect, DNA smears with a larger distribution before the
highest peak will report a smaller smear size compared to the peak size. Therefore, the most accurate sizing for a smear or library
sample can be achieved with the Smear Analysis tab on PROSize.

Abstract
Quality control check points for sizing are a necessity for many nucleic acid applications and ensures successful outcomes. The
Fragment Analyzer™ Automated CE System from Advanced Analytical Technologies, Inc. (AATI) provides reliable sizing for fragments
and smears with PROSize® Data Analysis Software. This application note describes factors that affect sizing and the best practices for
achieving accurate sizing of the sample.

A
LM

Peak Size
300 bp

Average Smear Size
312 bp

Introduction
Accurate sizing is essential for quality control analysis of nucleic acids. The Fragment Analyzer™ Automated CE System from Advanced
Analytical Technologies, Inc. (AATI) offers a wide range of qualitative and quantitative kits that provide reliable sizing for fragments
and smears with PROSize® Data Analysis Software. Standard deviation, precision, and accuracy were the parameters for evaluating
sizing. Standard deviation is used to quantify the amount of variation or dispersion of a set of data values from the mean. Accuracy
measures the closeness of a number to the true value and is reported as %error (Equation 1). Precision is the closeness of two or more
measurements and is independent of accuracy. A low %CV (coefficient of variance, Equation 2), representing precision, demonstrates
the closeness of the measurements and reliability of the average.
- known
( calculated
known

(

%Error =

* 100

Peak Size
350 bp

Size (bp)

%CV = std dev * 100
avg

Average Smear Size
488 bp

Size (bp)

Figure 1: Peak and smear size of DNA libraries analyzed on the Fragment Analyzer™ with the HS NGS Fragment Kit. (A) A DNA library with a bellshaped curve will report similar peak and smear sizes. (B) A DNA library with a larger distribution of fragments to the right of the highest peak will
have a larger smear size than peak size. Smear Analysis is recommended for sizing of all DNA smears and library samples.

Equation 2: Precision

Equation 1: Accuracy

Size (bp)

Size (bp)
LM

Sizing Under 6,000 bp Throughout a Dilution Series
Methods

The HS NGS Fragment Kit has a DNA sizing range of 100 - 6,000 bp.
Fragment sizes of 300 bp and 1,000 bp were analyzed on the
Fragment Analyzer with the HS NGS Fragment Kit throughout the
concentration range of the Kit (Figure 2A & B). The 300 bp and
1,000 bp fragments reported consistent sizing of 299 ± 1.24 bp
and 997 ± 3.35 bp respectively, with a %error below 0.3%. Both the
300 bp and the 1,000 bp fragments had excellent precision values
of 0.41% and 0.33% CV respectively, indicating consistent sizing
between the concentration points (Table 1).

Various fragment sizes - 300 bp, 1,000 bp, and 15,000 bp - were analyzed on the Fragment Analyzer with the DNF-474 High Sensitivity
NGS Fragment Analysis Kit (HS NGS Fragment Kit) and the DNF-464 High Sensitivity Large Fragment 50Kb Analysis Kit (HS Large
Fragment Kit) over a concentration range of 3.9 - 500 pg/µL and 4.5 - 600 pg/µL, respectively. A DNA smear was separated with
the DNF-477 High Sensitivity Small Fragment Analysis Kit (HS Small Fragment Kit) over a concentration range of 78 - 5,300 pg/µL.
The HS NGS ladder was diluted with 200, 100, 50, 25 mM NaCl or 1X TE buffer and separated on the Fragment Analyzer with the HS
NGS Fragment Kit.
Results & Discussion
Peak Size vs Smear Size

Average (bp)
Range (bp)
Std. Dev

300 bp
DNF-474

1,000 bp
DNF-474

2991

9971

297 - 302

992 - 1,001

1.2

3.3

% CV
0.41%
0.33%
The HS Small Fragment Kit has a DNA sizing range of 50 - 1,500 bp.
% Error
0.06%
-0.3%
A DNA smear was separated on the Fragment Analyzer with
Table
1:
Overview
of
sizing
data
for
300
bp
and
1,000
bp
fragments
the HS Small Fragment Kit over a concentration range of
over
a
concentration
range
separated
on
the
Fragment
Analyzer™
5,300 - 78 pg/µL (Figure 3A). The smear size remained consistent
with the HS NGS Fragment Kit. 1n=144.
at 295 bp, with no significant change over the entire concentration
range of the kit (Figure 3B). Both the Small Fragment Kits and NGS
Fragment Kits provide consistent fragment and smear sizing across the concentration range of the respective kits.

Fragments and smears have different separation profiles on electropherograms. Fragments are displayed as sharp peaks, while
smears are broader and lower in height than fragments with the same concentration. DNA libraries vary greatly in size and profile,
due to different modes of processing. They often have profiles similar to smears. Although, libraries with a small size range can display
a profile characteristic of both fragments and smears.
DNA size can be reported as a peak size or smear size. The peak size is the tallest/most concentrated portion of the sample, while the
smear size accounts for the entire distribution of the sample over the designated smear range. PROSize automatically reports a peak
size for all sample types, while the Smear Analysis tab in PROSize allows the user to set a base pair range for determination of the
average size and concentration of the smear. Peak size is recommended for sizing of DNA fragments.

Sizing Over 6,000 bp
The HS Large Fragment Kit has a DNA sizing range of 75 - 48,500 bp. To achieve the most reliable and accurate sizing with HS
Large Fragment Kit, an optimal fragment concentration range of 500 - 600 pg/µL and an optimal smear concentration of 1 ng/
µL is recommended. A 15,000 bp fragment was separated on the Fragment Analyzer with the HS Large Fragment Kit at the
recommended concentration of 600 pg/µL (Figure 4). An average size of 14,566 bp was reported with very high accuracy
(2.9% error) and precision (1.4% CV).

DNA smears were separated on the Fragment Analyzer with the HS NGS Fragment Kit in order to compare peak and smear sizing.
DNA smears or libraries with a typical bell curve or a uniform distribution on both sides of the highest peak of the sample, reported

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Sizing over Concentration Range
4.5 - 600 pg/µL

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Improved Nucleic Acid QC with Parallel CE

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