How To Recover Dna From Supernatant In Bead Clean Up

Office 2: Size Option for NGS libraries

SPRI beads

Magnetic beads are commonly used in molecular labs for purification of nucleic acids, but they have some other very important function in NGS workflows – size-selection.

Solid-Phase Reversible Immobilization (SPRI) beads were first introduced in 1998, and through the years have become an essential tool in high-throughput molecular labs for the purification of nucleic acids. They are predominately used in NGS workflows to make clean-up DNA or libraries during PCR purification which removes contaminants such as dNTPs, salts, primers and primer dimers. Due to their DNA binding backdrop, SPRI chaplet also allow for size-selection of DNA molecules which is a crucial step in NGS workflows to ensure the correct library size for sequencing.

There are currently many types of SPRI bead products on the market, even so, the AMPure XP chaplet from Beckman-Coulter are the most unremarkably used commercial version in South Africa (Table 1).

Production NAME	COMPANY
AMPure XP	Beckman Coulter Life Sciences
Mag-Demark TotalPure NGS	Omega Bio-tek
HigherPurity DNA Purification Chaplet	AG Scientific
ProNex	Promega
NucleoMag	Macherey-Nagel
PCRClean DX	Aline Biosciences

Table ane: Different commercial versions of SPRI beads on the market.

Bones principle of SPRI beads

SPRI beads are paramagnetic (merely magnetic in a magnetic field) particles which prevents them clumping together in solution. Each bead is fabricated of polystyrene surrounded past a layer of magnetite, which is coated with carboxyl molecules (Figure1). These carboxyl groups demark non-specifically and reversibly to Dna in the presence of polyethylene glycol (PEG) and salt which is independent in the liquid buffer they send in (twenty% PEG, two.5M NaCl). Both the salts and the PEG play a disquisitional role in forming DNA precipitate on magnetic chaplet. Salts provide ion bridging of negatively charged molecules such as DNA and SPRI beads, while PEG 'crowds' solutes and h2o-suspended substances together by shrinking water-soluble space. As the immobilization is dependent on the concentration of PEG and common salt in the reaction, the volumetric ratio of beads to DNA is important.

Figure 1: Composition of a SPRI bead particle

The standard procedure for a PCR purification is as follows (Figure 2):

1. Add together and mix 1.8 μL AMPure XP per 1.0 μL of sample (e.g. ninety μL beads to fifty μl sample).
2. Bind Deoxyribonucleic acid fragments to paramagnetic beads by incubating at RT for 5mins.
3. Separation of beads + Dna fragments from contaminants using a magnetic stand up. The supernatant containing the contaminants is aspirated and discarded.
4. Wash chaplet + Deoxyribonucleic acid fragments twice with 70% ethanol to further remove contaminants.
5. Elute purified DNA fragments from chaplet using molecular class water or TE buffer.
6. Transfer purified Dna solution to a new plate/tube.

Following these steps using a bead to sample ratio of 1.8x will bind all fragments greater than ~100 bp, leaving backside smaller fragments such as complimentary adapters and primers.

Figure 2: Standard workflow for the purification of a PCR reaction using SPRI beads (left-side selection)

Tips for treatment SPRI chaplet

• Thoroughly vortex beads before employ for at to the lowest degree 10 seconds.
• Store chaplet at the correct temperature (2 – 8°C).
• Allow enough time for beads to equilibrate to room temperature before utilize (~30mins).
• The bead solution is viscous, use a tedious pipetting technique and well-calibrated pipettes.

E'er prepare freshly diluted ethanol for wash steps.

Using SPRI Based Size-Selection in NGS

The ability to select for DNA fragments of a sure size range is an important step in all NGS workflows using short-read sequencing (e.g. Illumina and Ion Torrent) equally fragments that are likewise pocket-sized or too large will interfere with the sequencing chemical science. The size of fragments eluted from the chaplet (or that bind in the first place) is determined by the concentration of PEG, and this in turn is determined past the volume of beads to DNA.

Equally this ratio is changed the length of fragments binding and/or left in solution likewise changes, the lower the ratio of SPRI:Deoxyribonucleic acid the larger the fragments that bind the beads. Smaller fragments are retained in the buffer and are usually discarded resulting in unlike size ranges from a single sample with multiple purifications. This effect is due to DNA fragment size affecting the total charge per molecule, with larger fragments having larger charges, thus promoting the electrostatic interaction with the beads and displacing smaller fragments.

A standard make clean-upwards, as described to a higher place, will apply a dewdrop to sample ratio > ane.0 (normally ane.0x, i.2x or one.8x) which binds all fragments larger than 100 bp. This ensures maximum recovery of Deoxyribonucleic acid while removing the smaller unwanted fragments. Every bit the ratio is decreased, the smaller fragments are left in solution and larger fragments bind to the beads, for example, a ratio of 0.8x (i.e. xl μl beads to fifty μl sample) will bind all fragments larger than ~200 bp (Figure three). This is referred to equally a left-side size selection and it is important to remember that the DNA you are selecting for (large fragments) is leap to the chaplet while the supernatant contains the smaller fragments and is discarded. Practice not discard the beads before elution!

Figure 3: Left-side size pick – Bioanalyzer assay showing the result of decreasing ratios on fragment size spring to beads

Reverse to left-side is the right-side size selection which works via size exclusion (Figure 4). SPRI beads are mixed with DNA at the desired ratio to demark large fragments of a sure size, similar to the left-sided reaction. Although now the supernatant contains the smaller fragments you intend on keeping while the large fragments bound to beads are left behind and discarded. Don't discard the supernatant after initial binding! This method is likewise referred to as 'opposite' SPRI since you discard the beads and proceed the supernatant. The supernatant is then transferred to a fresh tube and a re-bind step is performed with fresh chaplet using ratios > 1.0 to commutation the buffer and select for all fragments larger than > 100 bp. When the resulting DNA fragments are analysed on a Bioanalyzer/ TapeStation, the fragment range observed for each ratio is the DNA which remained in the supernatant and not that which was initially bound to the beads and discarded (Figure 5)

Effigy 4: Right sided pick process showing the exclusion step where the big fragments remain bound to the beads and the supernatant is carried across to perform the re-bind step

Figure 5: Right-side size selection - the ratios to a higher place will leave the observed fragment size range in the supernatant which is then used to re-bind and capture all fragments larger than 100 bp

A combination of left- and right-sided size option tin be performed in several different ways, but the most common method is the same as the correct-side procedure described to a higher place except that it uses the re-bind step to perform a left-side option. This is called double-sided size selection and as a general rule, the left-side size choice ratio e'er needs to be greater than the right-side size pick ratio. The ability to manipulate the ratios in this way allows one to select for a tight size range, for example capturing all Dna fragments between 250 – 600 bp (Figure half dozen).

There is a formula to summate the volume of beads required for the left-side option rebind, subtract the Right-side ratio from the Left-side ratio, and multiply that by the initial book of sample: (Left Ratio – Right Ratio) * µL original sample.

Hither is a practical example:

Beginning bind (Right side ratio): 0.5x SPRI (48.5 µl beads, 97 µl DNA)

• Fragments >600 bp on beads.
• Fragments <600 bp in supernatant.
• Discard beads, proceed supernatant (0-600 bp).

Second bind (Left side ratio): 0.65x SPRI (fourteen.vi µL beads, 130 µL supernatant from previous step)

• Fragments >250 bp on beads.
• Fragments <250 bp in supernatant
• Discard supernatant, go on beads (250-600 bp)

The volume of beads required for the 2nd bind was calculated using the formulae to a higher place: (0.65-0.5)*97 = (0.15*97) = fourteen.6 µL

Figure six: Double-sided size choice showing the relationship betwixt fragment size range and the ratios used. Values for each graph testify left-side ratio and correct-side ratio.

As the region of Dna selected for becomes narrower there is a subtract in the corporeality of Dna binding from the original sample and therefore a decrease in the total amount of sample recovered (Table2). I must consider this potential sample loss when performing a double-sided size selection and make sure to start with enough input Deoxyribonucleic acid and so that enough is recovered for downstream processing.

Table 2: Recovery of Dna decreases as the size selection becomes narrower.

Definitions: Ratios (Left-Right): Left Side Size Selection ratio – Correct Side Size Selection ratio bp Region: Agilent 2100 Practiced Smear Region analyzed for the given Ratios (Left-Right) Selection Delta (bp): The difference between the Left and Right option points of the bp Region Example: 660 Right bp – 230 Left bp = 430 bp Delta

Alternatives to SPRI chaplet

Although dewdrop-based size selection is ideal for fragment sizes ranging from 100 – 800 bp, they are unable to efficiently bind large fragments over grand bp. Gel-based solutions have been used instead for targeting longer DNA fragments, for example, those required for long-read sequencing technologies such equally Pacific Biosciences (>6 Kbp) and Oxford Nanopore (>10 Kbp). Instruments such as the BluePippin from Sage Science use pulsed-field electrophoresis and have the capacity and flexibility to select for a large range of fragment sizes betwixt 100 bp – l 000 bp. Although effective at binding big fragments, gel-based size selection is express past long elapsing, expensive equipment, and poor sample recovery. A recent study was able to demonstrate that modifications to the SPRI bead buffer solution by adjusting the concentrations of salt and PEG were able to enhance the sizing threshold and allow selection of large DNA fragments up to 10 Kbp (Stortchevoi et al.).

Summary

SPRI chaplet can be used in a variety of NGS library prep chemistries and are compatible with both manual and automated processing. Their not-specific and reversible binding properties give scientists a tool for predictable and consistent size choice with high recovery of amplicons > 100 bp.

Contact u.s. on ross@diagnostech.co.za or michelle@diagnostech.co.za for more data and assistance with your NGS projects!

Author: Ross McFadyen

Ross joined Diagnostech in 2022 as a Field Application Scientist, supporting the Genomics portfolio. He has a keen interest in personalised medicine, particularly the apply of NGS in the clinical setting which he envisions becoming the standard for patients in the future as technological advances allow increased accessibility to these cutting-edge tools.

Source: https://diagnostech.co.za/next-generation-sequencing-tips-n-tricks-part-2/

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