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

In this application note, we demonstrate fully automated DNA size selection of 48 samples with CleanNGS magnetic beads on the ASSIST PLUS pipetting robot. The 8 channel 125 µl VOYAGER adjustable tip spacing pipette automates the liquid handling steps and the MAG module automates the magnetic bead handling steps in this protocol (we provide a semi-automated workflow using a magnet plate in the appendix).

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

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

Downloads: App note and protocols for single- and double-sided DNA size selection with the ASSIST PLUS pipetting robot

DNA single-sided size selection (PCR product purification)

Bind PCR product to a 1.8x magnetic bead ratio

Prepare fresh 80 % ethanol, and bring CleanNGS magnetic beads up to room temperature (RT). Place a 96 well DWP in portrait orientation on position A with 450 µl of CleanNGS magnetic beads in wells A1-A8 (Figure 2, light blue), 1.6 ml of 80 % ethanol in wells B1-B8 (Figure 2, blue) and 320 µl of molecular grade water in wells C1-C8 (Figure 2, pink). Place one empty 96 well HardShell PCR plate on position B and one with 40 µl of sample in each well of the first half in landscape orientation on the MAG module with the 96 well adapter on position C (Figure 2, yellow).

Select and run the VIALAB program 'MAG_PCR_product_purification'. With 125 µl sterile, filter, low retention GRIPTIPS®, VOYAGER on ASSIST PLUS will transfer 72 µl of magnetic beads from column A (Figure 2, light blue) of the DWP on position A (Figure 3a) to every well of the first half of the PCR plate on position C (Figure 3b). Magnetic beads are mixed 10 times before aspiration, and 15 times after dispensing into samples, guaranteeing a homogenous magnetic bead mixture. GRIPTIPS are changed automatically between samples. With the magnet array disengaged (Figure 4a, position home, 0 mm), VOYAGER will initiate an incubation of 5 minutes at RT to bind the PCR fragments to the magnetic beads (Figure 4b).

Removal of small fragments and washing

MAG will automatically lift the magnet array from position home (Figure 4b) to position high at 29 mm (Figure 5a) and capture magnetic beads within 3 minutes (Figure 5b). The PCR plate adapter for MAG has small holes to visually confirm targeted capturing of magnetic beads at the well surface.

VOYAGER – using fresh GRIPTIPS for each sample – removes the supernatant while the magnet array remains engaged (Figure 5a). The pipette transfers the supernatant into columns F-H of the DWP on position A. Slow aspiration (speed 1) and precise height settings prevent magnetic bead loss during washing. Magnetic beads are washed twice with 125 µl of 80 % ethanol from columns B of the DWP on position A (Figure 2, blue). VOYAGER aspirates an additional time to ensure complete removal of ethanol from each well. MAG will lower the magnet array by 5 mm (position 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 well bottom, 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.
• Worry-free liquid handling of ethanol uses fast aspiration and slow dispensing with a tip touch to prevent droplet formation.

Elution of single-sided size selected fragments

40 µl of molecular grade water is then transferred from column C of the DWP on position A (Figure 2, pink) to every well of the first half of the PCR plate on position C. Mixing 25 times ensures proper resuspension of the magnetic beads, independently of the volume, followed by a 5-minute RT incubation for elution. Again, MAG will automatically lift the magnet array to 29 mm (Figure 5a) and capture magnetic beads within 3 minutes (Figure 5b). Afterwards, VOYAGER transfers 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.

DNA double-sided size selection

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

The deck set-up for DNA double-sided size selection is similar to DNA single-sided size selection, but with 320 µl of CleanNGS magnetic beads (Figure 2, light blue), 350 µl of molecular grade water (Figure 2, pink), and 55 µl of sample in each well of the first half of a HardShell 96 well PCR plate (Figure 2, yellow).

Select and run the VIALAB program 'MAG_DNA_double_size_selection'. VOYAGER will follow the steps described in PCR product clean-up but transfers 38.5 µl of magnetic beads to each well containing a sample (Figure 3b). Mixing 10 times before every other aspiration using new GRIPTIPS before 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), VOYAGER will transfer 85 µl of supernatant from each well of the first half of the PCR plate to the corresponding well of the second half of the same plate on position C.

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

Following the same procedure as the right size selection, MAG lowers the magnet array back to position home (Figure 4a) and VOYAGER transfers 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 bead. The subsequent washing procedure mirrors the DNA single-sided size selection.

Elution of double-sided size selected fragments

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

Tip:

• Changing to different fragment sizes is trouble free, as the operator can calculate the magnetic bead volume for any ratio, and simply update it in VIALAB.

Results

Most reagent kit providers for library preparation recommend AMPure XP magnetic beads for NGS. Here, we demonstrate equivalent performance of CleanNGS magnetic beads during full automation of 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.

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

Figure 6 illustrates the gel picture of row A (CleanNGS) and row E (AMPure XP) of the 96 well plate when performing single-sided DNA size selection with 100 bp DNA ladder and a 1.8x magnetic bead ratio. Both magnetic beads 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 illustrates an electropherogram (EPG) with 22 out of 24 replicates (outliers excluded) of CleanNGS (left) and AMPure XP (right) when performing double-sided DNA size selection of sheared gDNA using 0.8x-0.7x (left-right) magnetic bead ratios. Both magnetic beads achieved a similar recovery of over 12 % (n=22) of 330 ng sheared gDNA. Average fragment sizes were 372 bp with CleanNGS and 396 bp with AMPure XP magnetic beads, while overall size variation for each reagent was below 5 %.

Remarks

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