DNA SIZE SELECTION
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 mostly benefit from the absence of short library fragments, whereas the short read sequencing technologies have a DNA insert size optimum between ca. 100 and 800 bp.
For most applications, the standard magnetic bead-based size selection is sufficient, however when performing miRNA sequencing experiments or if you like to limit the overlap between Read 1 and 2 in short read paired-end sequencing projects (eg. for de novo assembly), a more stringent type of size selection might be worthwile.
Following size selection, you might be interested in verifying the size distribution (DNA qualification) or to continue with Massive Parallel Sequencing.
METHODOLOGY
The principle of magnetic bead-based cleanups and size selection is based on Solid Phase Reversible Immobilization (SPRI). Here, nucleic acids (DNA/RNA) bind 'selectively' to carboxylated paramagnetic beads in a crowding agent (eg. PolyEthylene Glycol / PEG) in low-water conditions. The higher the water fraction, the more selective the magnetic beads become for high molecular weight DNA/RNA, so low molecular weight DNA/RNA remains in solution together with potential pollutants like cell-debris, salts, enzymes,... Using a magnetic field, the paramagnetic beads with the bound DNA/RNA get pulled out of the solution containing the contaminants. After removal of the supernatant and performing ethanol cleanup steps, the purified/size selected nucleic acids can be eluted in an aqueous solution.
Size selection using the Pippin prep is based on agarosis gel electrophoresis. Here, a user-defined DNA size-range is selected automatically by changing the current to a channel containing a collection well. Purified, size selected DNA can be pipetted out of this collection well and used for downstream applications.
Both principles of either the SPRI size selection/cleanup or Pippin Prep is visualized in the movies below.
Basic principle of magnetic bead-based cleanup (SPRI)
Overview of the Sage Science Pippin technology
INSTRUMENTS
The following methods are used for size selection in our premises.
Magnetic beads (multiple suppliers)
High throughput methodology to clean up nucleic acid samples and to (broadly) select for specific size ranges.
Sage Science Pippin Prep
Low throughput methodology for performing very tight DNA size selections, that are not possible using magnetic beads.
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.
Additional information can be found in the download section.
Assay metrics
Instrument
Sample type
Kits currently in use
Volume/input range
Throughput
Size range
Applications & remarks
Instrument
Applications (possible)
Applications (in use)
Remarks
Magnetic beads (multiple suppliers)
DNA/RNA cleanup, DNA/RNA size selection (broad)
DNA/RNA cleanup, DNA/RNA size selection (broad)
Indispensable methodology in any NGS/MPS setting : used for nucleic acid cleanups between subsequent and incompatible enzymatic manipulations. Broad size selections are possible, but it is hard to remove DNA fractions > 300 bp without dramatic loss of DNA.
As SPRI cleanups/size selections can be performed in 96 well plates, it is a high throughput method.
Due to the very high binding capacity of the beads (ca. 7 µg per µl beads) it is highly scalable.
Sage Science Pippin Prep
DNA size selection (tight and broad), miRNAseq, ChIPseq, Small genome sequencing (500-800 bp size selection)
DNA size selection (tight and broad), miRNAseq, Small genome sequencing (500-800 bp size selection)
This technology is best suited when very small fragments (eg. in miRNAseq applications) or very large fragments (eg. for small whole genome sequencing on an Illumina sequencer) are needed.
As only 5 samples/run can be processed, it is a low throughput method.
CERTIFICATES
Under validation
DOWNLOADS
Brochure Pippin family
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 |
|---|---|---|---|---|
NGS library (PCR free) | Microtube* or 96 well plate | See test details/specifications | Ice (max. 1 day) or dry ice | DNA |
NGS library (non-PCR free) | Microtube* or 96 well plate | See test details/specifications | Room temperature or ice pack | 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 |
|---|---|---|
Size selected/purified DNA | A tube/plate with size selected or purified DNA can be returned (according to your specifications) | RUO |
PLAN EXPERIMENT
To set experiments up with us, please follow the steps 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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...
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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 :
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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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...
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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
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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.
