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

In this application note, we demonstrate automated DNA size selection of 48 samples with CleanNGS magnetic beads using the 8 channel 125 µl VOYAGER adjustable tip spacing pipette on the ASSIST PLUS pipetting robot.

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

• Single-sided DNA size selection
• 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

Bind sheared gDNA 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 deep well plate (DWP) in portrait orientation on position A with 450 µl of CleanNGS magnetic beads in wells A1-A8 (Figure 2, light blue), 1.2 ml of 80 % ethanol in wells B1-B8 and C1‑C8 (Figure 2, blue) and 320 µl of molecular grade water in wells D1-D8 (Figure 2, pink). Place a 96 well skirted, low profile PCR plate with 40 µl of samples in each well of the first half in landscape orientation on position B (Figure 2, yellow) and the CleanNA magnet plate on position C.

Select and run the VIALAB program 'DNA_single_size_selection'. With 125 µl sterile, filter, low retention GRIPTIPS®, the 8 channel 125 µl VOYAGER adjustable tip spacing pipette on the ASSIST PLUS pipetting robot transfers 72 µl of magnetic beads (1.8x ratio) 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 B (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. Afterwards, the VOYAGER will initiate an incubation delay of 5 minutes at RT, so that DNA can bind to the magnetic beads.

Removal of small fragments and washing

The VOYAGER prompts the operator to place the PCR plate onto the magnet plate on Position C (Figure 4), followed by a 3 minute incubation to capture all magnetic beads with the ring magnet (Figure 5). When placing the PCR plate onto the magnet plate, the operator must ensure that the plate sits tight and straight by pressing down at the corners.

The VOYAGER – using fresh GRIPTIPS for each sample – removes the supernatant while the PCR plate remains on the magnet plate (Position C). The pipette transfers the supernatant into columns F-H of the DWP on position A. A slow aspiration (speed 2) and precise height settings prevent magnetic bead loss during washing. Magnetic beads are washed two times with 180 µl of 80 % ethanol from columns B and C of the DWP on position A (Figure 2, blue). The VOYAGER then aspirates the supernatant from each well again to ensure complete ethanol removal, followed by air drying for 3 minutes at RT.

Tip:

• Worry-free liquid handling of ethanol uses a 10 µl air gap after aspiration to prevent dripping. Dispensing is performed at a slow speed, followed by a tip touch at the side of the well to guarantee droplet removal.

Elution of single-sided size selected fragments

The operator is instructed to transfer the PCR plate from position C to B. 40 µl of molecular grade water is then transferred from column D of the DWP on position A (Figure 2, pink) to every well of the first half of the PCR plate on Position B (Figure 2, yellow). Mixing 15 times ensures proper re-suspension of the magnetic beads, followed by a 5 minute RT incubation for elution. The VOYAGER will then prompt the operator to place the PCR plate back onto the magnet plate on position C (Figure 4), and to place a new 96 well PCR plate on position B. After a 3 minute delay to capture magnetic beads with the ring magnet (Figure 5). The VOYAGER transfers 35 µl of eluate to the new 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 informed to store the PCR plate from position B, and remove the plate from the magnetic plate.

DNA double-sided size selection

Binding sheared gDNA to a 0.7x magnetic bead ratio

The deck set-up for DNA double-sided size selection is similar to single-sided DNA 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 'DNA_double_size_selection'. The VOYAGER will follow the steps described in single-sided DNA size selection, but transfers 38.5 µl of magnetic beads to each well containing samples (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) with the ring magnet (Figure 5), the VOYAGER prompts the operator to place a new HardShell 96 well PCR plate on position B. It then transfers 85 µl of supernatant from each well in position C to the corresponding well in the first half of the new PCR plate. Before starting the second DNA size selection, the VOYAGER informs the operator to remove the plate from the Clean magnet plate.

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

Following the same procedure as the right size selection, the VOYAGER transfers 5 µl of magnetic beads to the supernatant of the first size selection. A 5 µl pre-dispense guarantees accurate pipetting of small volumes of magnetic bead. The subsequent washing procedure mirrors single-sided DNA size selection, with a 5 minute delay to capture magnetic beads and prevent any bead loss.

Elution of double-sided size selected fragments

The VOYAGER follows the same procedure as the DNA single-sided size selection but transferring 50 µl of molecular grade water before elution, and 45 µl after capturing magnetic beads with the ring magnet.

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 higher reproducibility and equivalent performance of CleanNGS magnetic beads in DNA size selection, showing the interchangeability while reducing the experimental costs. Therefore, we performed single- and double-sided DNA size selection of sheared gDNA with CleanNGS magnetic beads while simultaneously comparing them to AMPure XP magnetic beads using the VOYAGER adjustable tip spacing pipette on the ASSIST PLUS pipetting robot.

We processed 48 replicates using AMPure XP magnetic beads in rows A to D and CleanNGS magnetic beads in rows E to H. The size-selected fragments (CleanNGS vs. AMPure XP) were analyzed and compared using the 4150 TapeStation System (Agilent, complete data can be found in the appendix). Figure 6 illustrates the electropherograms of (a) single-sided and (b) double-sided DNA size selection for row A (AMPure XP) and row E (CleanNGS).

Both reagents effectively removed fragments smaller than 100 bp during single-sided size selection using a 1.8x magnetic bead ratio. The average yield of sheared gDNA processed with CleanNGS magnetic beads was 5.5 ng/µl (±0.25) and 6.0 ng/µl (±0.45) with AMPure XP magnetic beads (Figure 6a; 6 out of 24 replicates shown per magnetic bead; left = Row E CleanNGS; right = Row A AMPure XP).

Double-sided DNA size selection with 0.8x-0.7x (left-right) magnetic bead ratios yielded average fragment sizes of 358 bp (±9.9) with CleanNGS and 371 bp (±10.4) with AMPure XP magnetic beads. Both reagents achieved a recovery of over 12 %. (Figure 6b; 6 out of 24 replicates shown per magnetic bead; left = Row E CleanNGS; right = Row A AMPure XP).

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.