DNA FRAGMENTATION

ABOUT

 

Massive Parallel Sequencing (MPS) requires input DNA fragments of specific sizes, where the size depends on (1) the sequencing technology and (2) the application. Long read sequencing technologies require no or minimal fragmentation whereas the short read sequencing technologies have a DNA insert size optimum between ca. 100 and 800 bp.

Next to enzymatic DNA fragmentation - as applied in various library preparation methodologies - mechanical DNA fragmentation may be preferred. The mechanical, ultrasonication based devices are considered as the gold standard due to their  'randomness' as compared to a more site-directed fragmentation with enzymes. Also, using ultrasonic fragmentation, (1) it is easier to optimize the fragmentation, (2) it is less sensitive to GC content and (3) can tolerate a larger DNA-input range. On the other hand, enzymatic fragmentation has the benefit of (1) scalability on the level of input volume and number of samples, (2) ease of use / time required, (3) reduced risk of sample loss or mix-ups as it is not needed to transfer samples to specific tubes and (4) a lower cost.

To assess DNA/protein interactions (eg. via ChIPseq), an ultrasonic fragmentation is the default methodology.

Following fragmentation, you might be interested in verifying the size distribution (DNA qualification) or to continue with Massive Parallel Sequencing.

METHODOLOGY

 

The principle of both the Adaptive Focused Acoustics (AFA) used by Covaris and ultrasonic fragmentation used by Diagenode is the same. Both reside on - via ultrasonic waves - the formation of bubbles (cavitation) and subsequent collapse/implosion of these bubbles, creating mechanical stress on the sample of interest. These lateral forces can fragment DNA, but also disrupt or homogenize tissues.

The AFA technology directs its power to a specific point in a microtube, whereas the ultrasonic method of Diagenode disperses its energy in a water bath. By rotating the microtubes in the water bath, each tube/sample gets an equal amount of energy. The Diagenode methodology is considered to be 'more gentle' than the AFA technology and it requires less costly consumables. Both devices perform very similarly, but the Covaris has a slightly larger range in the DNA fragment size.

Other fragmentation technologies exist : (1) the frequently used enzymatic method (can be restriction enzymes, nicking enzymes or transposases), (2) chemical fragmentation eg. by using divalent cations and heat, (3) hydrodynamic shearing, where the liquid sample is pushed through a small nozzle/syringe and (4) nebulization where compressed air is used to push the sample throuh a nebulizer (nozzle).

The methodology of the AFA technology is visualized in the movie below.

Overview of the Adaptive Focused Acoustics (AFA) as used by Covaris

 

INSTRUMENTS

The following devices are used for DNA/RNA fragmentation in our premises.

Covaris M220

Low throughput DNA fragmentation device (single tube). High quality fragmentation results, but costly tubes required.

Diagenode BioRuptor Pico

Medium throughput (6 - 12 samples) DNA fragmentation device resulting in high quality fragmentation results using low cost plastics.

SPECIFICATIONS

 

The specifications are subdivided in 2 sections : (1) the assay metrics and (2) applications & remarks.

More information can be found on the supplier page through the corresponding buttons in the 'Assay metrics' section. 

The link to DNA shearing instructions can be found in the download section.

Assay metrics

Instrument

Sample type

Tubes currently in use

Volume/input range

Throughput

Size range

Covaris M220

DNA/chromatin

microTUBE-15
microTUBE-50

150-550 bp*

15-20 µl** ; ≤ 50 ng/µl³
55 µl** ; ≤ 100 ng/µl³

1 sample / run

Diagenode BioRuptor Pico

DNA/chromatin

0,65 ml microtubes
1,5 ml microtubes

150-1000 bp
200 bp

100 µl² ; ≤ 100 ng/µl³
100-300 µl² ; ≤ 100 ng/µl³

12 samples / run
6 samples / run

* Other size ranges are possible using other tibes (eg. miniTUBES can generate sizes between 2 and 5 kb)

** A ca. 5% deviation in the input volumes are allowed for

² Using 0.2 ml microtubes, it is possible to perform fragmentation experiments in input volumes of 20-100 µl

³ Preferred solvent is TE buffer (10 mM Tris, 1 mM EDTA, pH 7.5 - 8.0) or Low TE (10 mM Tris, 0.1 mM EDTA, pH 7.5 - 8.0)

Applications & remarks

Instrument

Applications (possible)

Applications (in use)

Remarks

Covaris M220

DNA/RNA/chromatin shearing, FFPE nucleic acid extraction, Cell lysis, Tissue disruption, Protein extraction from cells and tissues, DNA extraction from Dried Blood spots (DBS)

DNA/RNA/chromatin shearing

High quality and consistent fragmentation results and - using the miniTUBES - has the potential to generate fragments ranging from 2000 to 5000 bp. Currently, mostly used for the TruSight Oncology 500 (TSO500) experiments.

Diagenode BioRuptor Pico

DNA/RNA/chromatin shearing, FFPE nucleic acid extraction, Cell lysis, Tissue disruption, Protein extraction from cells and tissues

DNA/RNA/chromatin shearing

Lowest operational cost while generating high quality and consistent fragmentation results. Our preferred instrument for e.g. ChIPseq experiments.

 

CERTIFICATES

Under validation
Under validation

DOWNLOADS

 
Flyer2 GW testing white borders_edited.j
DNA Shearing with BioRuptor Pico

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Flyer2 GW testing white borders_edited.j
DNA Shearing with M220 Focused-ultrasonicator

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CONTACTS

 
pas14klein.jpg

BRIGHTcore general inquires

+32 (0)2 477 64 79 (sec)

UZ Brussel

BRIGHTcore

Laarbeeklaan 101

1090 Brussels

SAMPLE TYPES

 

We aim to keep te list with sample types updated. However, if you believe we offer this test on other sample types, of if you have a very specific sample type you'd like to evaluate : please contact us.

Tissue
Recipient
Quantity
Transport
Purpose / Extract
DNA from various tissues
Microtube* or 96 well plate
See test details/specifications
Room temperature
DNA

* Microtube can be  a either a cryovial or eppendorf tube (0,5 ; 1,5 or 2 ml). Please consider the quality of the tube used (nuclease free ; free of RNA/DNA ; sterile ; LoBind) according to application needed.  

 

DELIVERABLES

Below, you can find the deliverable for this test.

If you require more information or need a more custom output, please contact us.

Deliverable
Description
Research or diagnostic
Fragmented DNA / chromatin
A tube with fragmented DNA/chromatin can be returned (according to your specifications)
RUO
 

PLAN EXPERIMENT

To set experiments up with us, please follow the steps below.
1. If you were trained to use our fragmentation devices
2. If you weren't trained to use the device
  • Please use the guidelines below

Get in touch (ONLY FOR NEW PROJECTS)

Explain your project, so we can assist you to find the best solution.

Expect questions like :

  1. What type of sample would you like to analyze?

  2. What is the aim of your experiment?

  3. How many samples would you like to process?

  4. How fast do you need the results (TAT) and/or do we need to expedite?

  5. If you deviate from our sample types, are there test samples available?

  6. Are our standard deliverables ok?

  7. ...

Call Center Headset

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Request quotation

Once you know which experiment you want to set up, fill out the 'Quote request' form, with the essential details of your project :

  1. Coordinates of the person to whom to address the quotation

  2. Which type of test you'd like to set up

  3. Do you require a specific deliverable?

  4. Do we need to expedite (comes at extra cost)?

  5. ...

Image by Andre Taissin

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Submit your samples

Once you agree with our terms (quote / TAT / terms & conditions)  :

  1. Fill out our sample submission form

    1. Send it via email : contact us

    2. Print out the form and include it with your samples 

  2. Send your samples to us​​

    1. Follow the instuctions given during the "Get in touch (1)" phase

    2. Label your tubes/plates correctly : see details here

    3. Adhere to the corresponding transport conditions listed under Sample types

  3. Indicate if we can discard your samples​ after completion of project

Blood Samples

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Your experiment starts

Now it is up to us... We will start your experiment as soon as possible.

  1. When expedited, the turn-around-time (TAT) is 2 workdays

  2. For standard requests the turn-around-time (TAT) is 5 workdays

The standard TAT is generally overestimated, but this extended timeframe allows us to : combine similar requests of multiple scientists and to repeat the experiment in case of issues. 

Please refrain from contacting us in case the pre-agreed TAT didn't expire yet.

Alarm Clock

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