Overview: How to automate DNA size selection with the ASSIST PLUS and MAG module

This application note describes the fully automated DNA size selection of 48 samples with MAGFLO NGS beads on the ASSIST PLUS pipetting robot. An 8 channel 125 μl VOYAGER automates the liquid handling steps, and the MAG module automates the magnetic bead handling steps.

Figure 1 illustrates the step-by-step procedure of the provided NGS size selection protocols for:

• Single-sided DNA size selection (PCR product purification)
• Double-sided DNA size selection

Downloads: App note and protocols for automated DNA size selection for flexible NGS integration

Experimental set-up

Single-sided DNA size selection (PCR product purification)

Binding the PCR product using a 1.8x magnetic bead ratio

Prepare fresh 80 % ethanol and bring MAGFLO NGS beads to room temperature (RT). Place a 96 well INTEGRA DWP on Position A of the ASSIST PLUS in portrait orientation, with 450 μl of MAGFLO NGS beads in wells A1-A8 (Figure 2, lilac), 1.6 ml of 80 % ethanol in wells B1-B8 (Figure 2, green) and 320 μl of molecular-grade water in wells C1-C8 (Figure 2, pink). Place one empty 96 well Bio-Rad Hard-Shell PCR plate on Position B. Place a second 96 well plate containing 40 μl of sample in each well in the first half (Figure 2, blue), in landscape orientation on the 96 well adapter of the MAG module on Position C.

Select and run the VIALAB program 'MAG_PCR_product_purification'. Using 125 μl sterile, filter, low retention GRIPTIPS®, the VOYAGER on the ASSIST PLUS will transfer 72 μl of magnetic beads from column A (Figure 2, lilac) of the INTEGRA DWP on Position A (Figure 3a) to each well in the first half of the PCR plate on Position C (Figure 3b). Magnetic beads will be mixed 10 times before aspiration, and 15 times after dispensing into samples, to guarantee a homogeneous magnetic bead mixture. GRIPTIPS will be changed automatically between samples. With the magnet array disengaged in Position Home (Figure 4a, Pos. Home, 0 mm), the VOYAGER will initiate an incubation of 5 minutes at RT, binding the PCR fragments to the magnetic beads (Figure 4b).

Removal of small fragments and washing

The MAG module will automatically lift the magnet array from Pos. Home (Figure 4b) to Position High (Pos. High) at 29 mm (Figure 5a) for 3 minutes to capture the magnetic beads (Figure 5b). The PCR plate adapter for the MAG module has small holes to visually confirm the targeted capture of magnetic beads at the well surface.

The VOYAGER – using fresh GRIPTIPS for each sample – will remove the supernatant while the magnet array remains engaged (Figure 5a). The pipette will then transfer the supernatant into columns F-H of the INTEGRA DWP on Position A. Slow aspiration (Speed 1) and precise height settings prevent magnetic bead loss. Next, magnetic beads will be washed twice with 125 μl of 80 % ethanol from column B of the INTEGRA DWP on Position A (Figure 2, green). The VOYAGER will aspirate an additional time to ensure the complete removal of ethanol from each well. The MAG module will then lower the magnet array by 5 mm to Position Low (Pos. Low, 24 mm) followed by air drying for 3 minutes at RT. Lowering the magnet array before air drying will move the pellets closer to the bottom of the wells, allowing easier elution and smaller volumes.

Tips:

• The magnet step in VIALAB provides total control of the magnet array by setting customized heights anywhere between 0 and 29 mm.
• When handling ethanol, fast aspiration and slow dispensing with a tip touch are required to prevent droplet formation.
• The drying condition has been optimized to 3 minutes, but may vary in different lab conditions.

Elution of single-sided size selected fragments

40 μl of molecular-grade water will be transferred from column C of the INTEGRA DWP on Position A (Figure 2, pink) to every well in the first half of the PCR plate on Position C. Mixing 25 times ensures proper resuspension of the magnetic beads, regardless of volume. This is followed by an incubation at RT for 5 minutes. Again, the MAG module will then automatically lift the magnet array to 29 mm (Figure 5a) for 3 minutes to capture the magnetic beads (Figure 5b). Afterwards, the VOYAGER will transfer 35 μl of eluate to the unused PCR plate on Position B, leaving 5 μl in the plate at Position C to prevent magnetic bead carryover. At the end of the run, the user is prompted to store the PCR plate from Position B, and remove the plate from the MAG module.

DNA double-sided size selection

Binding sheared genomic DNA (gDNA) using a 0.7x magnetic bead ratio

The deck set-up for double-sided DNA size selection is similar to single-sided size selection, but with 320 μl of MAGFLO NGS beads (Figure 2, lilac), 350 μl of molecular-grade water (Figure 2, pink), and 55 μl of sample in each well in the first half of a Bio-Rad Hard-Shell 96 well PCR plate (Figure 2, blue). 

Select and run the VIALAB program ‘MAG_DNA_double_size_selection’. The VOYAGER will follow the steps described in PCR product purification, but will transfer 38.5 μl of magnetic beads to each well containing a sample (Figure 3b). Mixing 10 times before every other aspiration – and using new GRIPTIPS before each aspiration – guarantees precise, low volume pipetting of magnetic beads.

Removal of large fragments (right size selection)

After capturing large fragments bound to magnetic beads (right size selection) (Figure 5b), the VOYAGER will transfer 85 μl of supernatant from each well in the first half of the PCR plate to the corresponding well in the second half of the same plate on Position C.

Binding target fragments using a 0.8x magnetic bead ratio, and removing small fragments (left size selection) during the washing process

Following the same procedure as the right size selection, the MAG module will lower the magnet array back to Pos. Home (Figure 4a), then the VOYAGER will transfer 5 μl of magnetic beads to the supernatant of the first size selection in the second half of the PCR plate (Position C). A 5 μl pre-dispense guarantees accurate pipetting of small volumes of magnetic beads. The subsequent washing procedure mirrors the single-sided DNA size selection.

Elution of double-sided size selected fragments

The MAG module and the VOYAGER will follow the same procedure used for single-sided DNA size selection, but 50 μl of molecular-grade water will be transferred before elution, and 45 μl after capturing the magnetic beads (Figure 5b).

Tip:

• VIALAB software enables operators to switch to different fragment sizes as needed. Simply calculate the magnetic bead volume for any ratio, and update it in VIALAB.

Results

Most providers of reagent kits for NGS library preparation recommend AMPure XP magnetic beads. This application note demonstrates the equivalent performance of MAGFLO NGS magnetic beads during automated single-sided DNA size selection – using a 100 bp DNA ladder (Promega) to mimic PCR product purification – and double-sided DNA size selection, using sheared gDNA.

Using the VOYAGER on the ASSIST PLUS, 48 replicates were processed with MAGFLO NGS beads in rows A to D, and AMPure XP beads in rows E to H. Automated magnetic bead handling with optimized magnet array heights was ensured by using the MAG module. The size-selected fragments were analyzed and compared using the 4150 TapeStation System (Agilent, complete data set can be found in the appendix).

Figure 6 shows the gel picture of row A (MAGFLO NGS beads) and row E (AMPure XP beads) of the 96 well plate for single-sided DNA size selection with a 30-fold diluted 100 bp DNA ladder and a 1.8x magnetic bead ratio. Both reagents purified all fragments of PCR ladder smaller than 100 bp and ~70 % of 4 ng 100 bp fragments, while recovering ~100 % of 65 ng fragments ranging from 200 bp to 1500 bp.

Figure 7 depicts an electropherogram (EPG) showing 22 out of 24 replicates (outliers excluded) for MAGFLO NGS (left) and AMPure XP (right) beads during double-sided DNA size selection of sheared gDNA. Magnetic bead ratios of 0.8x and 0.7x were used for left and right size selection, respectively. Both reagents achieved similar recovery rates, exceeding 12 % (n=22) of 330 ng sheared gDNA. Average fragment sizes were 372 bp with MAGFLO NGS beads, and 396 bp with AMPure XP beads, with overall size variation for each reagent remaining below 5 %.

Remarks

• VIALAB software: The VIALAB programs can be easily adapted for specific pipettes, labware and protocols.
• Partial plates: Pre-set programs offer laboratories complete flexibility to accommodate varying sample sizes, ensuring they can
  meet both current and future demands.
• Semi-automation: The appendix includes a protocol that uses a magnet plate for a semi-automated workflow.