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

In this application note, we demonstrate the fully automated DNA size selection of 48 samples with MAGFLO NGS – magnetic beads for NGS size selection – 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. A Quick start guide, a concise document designed to help operators quickly set up the application, is available for download in the section below.

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 automated DNA size selection for flexible NGS integration

DNA single-sided size selection (PCR product purification)

Bind PCR product to a 1.8x magnetic bead ratio

Prepare fresh 80 % ethanol and bring MAGFLO NGS to room temperature (RT). Place the 96 well INTEGRA DWP in portrait orientation on position A with 450 µl of MAGFLO NGS 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 1 empty 96 well Bio-Rad Hard-Shell PCR plate on position B and 1 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, blue).

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, lilac) of the INTEGRA DWP on position A (Figure 3a) to every well of 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, guaranteeing 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), 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 Pos. Home (Figure 4b) to position high (Pos. 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 the targeted capturing of magnetic beads at the well surface.

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 during washing. Magnetic beads will then be washed twice with 125 µl of 80 % ethanol from column B of the INTEGRA DWP on position A (Figure 2, green). VOYAGER will aspirate an additional time to ensure the complete removal of ethanol from each well. MAG will 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 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.
• 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 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 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 magnetic plate.

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 MAGFLO NGS (Figure 2, lilac), 350 µl of molecular grade water (Figure 2, pink), and 55 µl of sample in each well of 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'. VOYAGER will follow the steps described in PCR product clean-up but will transfer 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 will lower the magnet array back to Pos. Home (Figure 4a) and 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 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 the equivalent performance of MAGFLO NGS – our magnetic bead for NGS size selection – 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 MAGFLO NGS 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 (MAGFLO NGS 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 (MAGFLO NGS) and row E (AMPure XP) of the 96 well plate when performing single-sided DNA size selection with 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 illustrates an electropherogram (EPG) with 22 out of 24 replicates (outliers excluded) of MAGFLO NGS (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 reagents achieved a similar recovery of over 12 % (n=22) of 330 ng sheared gDNA. Average fragment sizes were 372 bp with MAGFLO NGS and 396 bp with AMPure XP magnetic beads, while the 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