Agilent 2019 - 8

Automated Nucleic Acid Sample Quality Control in NGS Workflows

Characterization of sheared DNA

8

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120
100
80
60
40
20

1,500

700

500

400

300

200

150

Size (bp)

1,
00
Up 0
pe
r

Lo

×103
16

10
0

w

er

100

0

B

14
12
10
8
6
4
2

1,000
1,500

700

500

400

300

200

100

50

0
25

Due to the modified shearing protocol, the
expected average size range of fragmented DNA
is about 100 bp larger than recommended in the
protocol (between 200 and 325 bp). The fragmented
DNA samples showed a maximum peak size
between 260 and 310 bp on both systems, which
verified optimal shearing. The size of this QC step
can be used as a core value for each sample to
compare with the size after adapter ligation later
in the SureSelectXT workflow. The sizing results of
the 2100 Bioanalyzer and the 4150 TapeStation

A

15
50

Example electropherograms of sheared DNA
analyzed with the 4150 TapeStation and the 2100
Bioanalyzer systems are shown in Figure 2. All
samples displayed an even size distribution with
no undesirable shouldering.

FU

Sample intensity (normalized FU)

The first step of the SureSelectXT protocol is
fragmentation of gDNA by shearing with the
Covaris ultrasonicator. The shearing protocol was
adjusted to produce fragments with a larger target
size to allow for sequencing in 2 × 100 bp mode.
Optimal shearing in NGS workflows is typically
verified with the evaluation of the size distribution
and electropherogram pattern of fragmented DNA
samples with the DNA 1000 assay and the 2100
Bioanalyzer system. In addition, the samples were
analyzed with the 4150 TapeStation system and
the D1000 ScreenTape assay.

Size (bp)

Figure 2. A fragmented DNA sample from the SureSelectXT
workflow showing even size distribution. A) Sheared
DNA analyzed with the Agilent DNA 1000 assay,
B) Electropherogram of the same sample analyzed with
the Agilent D1000 ScreenTape assay.



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Agilent 2019

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