PROTOCOLDNA shearing for Next-Generation Sequencing
with the Bioruptor® PicoOptimal data generation using NGS platforms relies on a few sample-preparation prerequisites, such as precise
DNA fragmentation. The Diagenode Bioruptor Pico ensures that DNA is efficiently sheared to an appropriate andconsistent fragment size. This first step is critical to generate high quality unbiased libraries.

DNA shearing using the Bioruptor Pico• allows simultaneous shearing from 6 up to 16 samples• compatible with a volume range from 20 μl up to 300 μl per sample• compatible with a broad range of sample concentrations• provides unbiased shearing and high yields of dsDNAA

B 200 bp

C 350 bp

D 550 bp

Programmable DNA size distributions, excellent reproducibility, and high dsDNA yields with the Bioruptor Pico.
Figure A shows different DNA size distributions of sheared genomic DNA produced by varying the duration ofsonication. The different curves depict a specific Bioruptor Pico run, optimized to produce specific mean sizes
and size ranges for NGS. Figures B-D show the excellent reproducibility in DNA shearing compatible with NGSlibraries from Illumina, IonTorrent and exon capture protocols from Agilent and Roche NimbleGen requirements.
All DNA samples were analysed on Fragment Analyzer™ (Agilent).

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www.diagenode.com Please contact us for more information
Europe Diagenode sa / LIEGE SCIENCE PARK // Rue du Bois Saint-Jean, 3 // 4102 Seraing (Ougrée) // Belgium // Phone: (+32) 4 364 20 50 // E-mail: info@diagenode.com
USA Diagenode Inc. / 400 Morris Avenue, Suite 101 // Denville, NJ 07834 // USA // Phone: (+1) 862 209-4680 // Mail: info.na@diagenode.com

Tube Holders and Tubes for Bioruptor Pico recommended for DNA shearing
The holder is made up of a tube holder and a specific dock (see the table below). Each tube holder accommodates
specific microtubes allowing simultaneous processing of multiple samples with variable sample volume as
indicated in the table below.

Tube Holder

Tubes

Adaptor for 0.65 ml tubes Bioruptor® Pico holder 0.65 ml Bioruptor® Microtubes(B01201143)(C30010011)

Samplevolume*# of samples
per run

100 μl12Adaptor for 0.2 ml tubes Bioruptor® Pico holder(B01201144)

0.2 ml microtubes for Bioruptor Pico(C30010020)

20-100 μl

16Adaptor for 1.5 ml tubes Bioruptor® Pico holder(B01201140)

1.5 ml microtubes for Bioruptor Pico(C30010016)

100-300 μl

6The sonication settings listed below are recommended guidelines for the shearing of genomic DNA of highmolecular weight. To ensure reproducible and consistent shearing, use pure RNA-free genomic DNA. Actual
results may vary depending on starting material, purity level, etc. Some additional optimization may be required
to determine the appropriate treatment for your specific sample. Please note also that referred sizes can differ
depending on the analytical systems used for the size assessment.
The successful shearing of FFPE-derived DNA relies on the initial quality and size of DNA. If extracted
DNA is of high molecular weight above 10 kb, the standard DNA shearing protocols should be applied.
Smaller DNA fragments are more resistant to sonication and the total sonication time should be extended
by additional cycles to reach the target size. Time course monitoring after 5, 10, 15 additional cycles isrecommended. Please refer for more details on FFPE-derived DNA shearing to the application note:https://www.diagenode.com/files/application_notes/AN-FFPE-07_2017.pdf
Shearing of PCR amplicons of the size down to 500 bp can be achieved using the Bioruptor Pico. PCR fragments
bigger than 700 bp are sheared according to the standard protocol, smaller PCR fragments from 500 - 700 bp
are less susceptible to sonication. For fragments of this size range the sonication time has to be increased byadding 3 to 5 additional cycles to the standard protocol.

2PROTOCOL
1.

Switch on the Bioruptor Pico and set the temperature of the cooler at 4°C.

2.

 repare the DNA solution at concentration 2-100 ng/μl* using TE buffer (10 mM Tris, 1 mM
PEDTA, pH 7.5 - 8.0) or Low TE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.5 - 8.0).

*NOTE: The smear of a sample with a concentration below 2 ng/µl will not be detected by most QC
instruments.
3.

 ransfer a required volume of DNA solution to the appropriate sonication microtubes accordingly to volumeTspecification.

4.

Briefly vortex (5-10 sec) and centrifuge (10 sec) samples.

5.

Incubate on ice for 10 min.

6.

Fill the tube holder with sonication tubes.

NOTE: To guarantee homogeneity of shearing, always completely fill the tube holder with tubes. Neverleave empty spaces in the tube holder. Fill the empty spaces with tubes containing the same volume of
distilled water.
7.

 onicate samples using the Bioruptor Pico. The mode of use, sonication cycle & total sonication time varies
S
depending on accessories and consumables, desired DNA size and initial sample volume. Please choose
appropriate settings from the tables below.

Please note that referred sizes have been assessed using the Fragment Analyzer™ from Agilent (smear analysisoption). They may differ depending on the analytical systems used for the size assessment.

Sonication settings for 0.2 ml tubes Bioruptor Pico holder and 0.2 ml Bioruptor®
Microtubes
The minimum recommended volume per sample is 20 μl.
Sonication settings for sample volume between 20 and 50 should be optimized by the operator using settingsfor 50 μl as a guideline.
Sonication settings for sample volume <50 μl -100 μl should be optimized by the by the operator using settingsfor 100 μl as a guideline.

20 μL SAMPLE VOLUME
target size, bp

Cycle number

Cycle conditions (On/Off time)

1000

315/30650

515/30450

530/30300

1030/30250

1530/30

2003030/30

Mode of use

Advanced mode:
ultra-low frequency

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50 μL SAMPLE VOLUME
target size, bp

Cycle number

Cycle conditions (On/Off time)

600

315/30450515/30350

430/30300

630/30200

1330/30150

2330/30

Mode of use

Easy mode:
0.2 ml tubes100 μL SAMPLE VOLUME
target size, bp

Cycle number

Cycle conditions (On/Off time)

1000

315/30650

515/304501030/30350

1530/30250

2030/30

200

30

30/30

Mode of use

Easy mode:
0.2 ml tubes

Sonication settings for 0.65 ml tubes Bioruptor Pico holder and 0.65 ml Bioruptor®
Microtubes
We highly recommend using 100 μl sample volume when working with 0.65 ml tubes. Any deviation will lead topoor shearing and the lack of the reproducibility.

100 μl SAMPLE VOLUME
target size, bp

Cycle number

Cycle conditions (On/Off time)

1000130/30

600

230/30400

530/30350

630/30200

1330/30

1802530/30

1503030/30

Mode of use

Easy mode:
0.65 ml tubes

4Sonication settings for 1.5 ml tubes Bioruptor Pico holder and 1.5 ml Bioruptor®
Microtubes
300 μL SAMPLE VOLUME
target size, bp

Cycle number

Cycle conditions (On/Off time)

Mode of use

200

1330/30

Easy mode:
1.5 ml tubes*- Sonication settings to achieve other target sizes and/or other sample volumes should be optimized by user.

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