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 through 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
* Other size ranges are possible using other tubes (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
DOWNLOADS
DNA Shearing with BioRuptor Pico
1
DNA Shearing with M220 Focused-ultrasonicator
1
CONTACTS
BRIGHTcore general inquires
+32 (0)2 477 64 79 (sec)
UZ Brussel
BRIGHTcore
Laarbeeklaan 101
1090 Brussels
SAMPLE TYPES
We aim to keep the list with sample types updated. However, if you believe we offer this test on other sample types, or 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
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Book the timeslot required in our booking tool
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Provide us with the list of samples (digitally) you've processed on the device
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Indicate whether or not you required consumables from us on the sample submission form
2. If you weren't trained to use the device
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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 :
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What type of sample would you like to analyze?
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What is the aim of your experiment?
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How many samples would you like to process?
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How fast do you need the results (TAT) and/or do we need to expedite?
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If you deviate from our sample types, are there test samples available?
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Are our standard deliverables ok?
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...
1
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 :
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Coordinates of the person to whom to address the quotation
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Which type of test you'd like to set up
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Do you require a specific deliverable?
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Do we need to expedite (comes at extra cost)?
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...
2
Submit your samples
Once you agree with our terms (quote / TAT / terms & conditions) :
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Fill out our sample submission form
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Send it via email : contact us
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Print out the form and include it with your samples
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Send your samples to us
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Follow the instuctions given during the "Get in touch (1)" phase
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Label your tubes/plates correctly : see details here
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Adhere to the corresponding transport conditions listed under Sample types
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Indicate if we can discard your samples after completion of project
3
Your experiment starts
Now it is up to us... We will start your experiment as soon as possible.
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When expedited, the turn-around-time (TAT) is 2 workdays
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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.
