Michael Farrell1, Henry She1, Travis Butts1, Jeffrey Roeder1 and Keith McKenna2.
1Omega Bio-tek, Norcross, GA, 2Alpaqua Engineering, Beverly, MA
Omega Bioservices supports its full suite of laboratory services by establishing customized automated workflows on open platform liquid handlers such as the Hamilton Microlab STAR. One of the newest services provided by Omega Bioservices is high throughput gut microbiome population identification from stool samples through 16s rRNA gene sequencing. Creating this high throughput automated workflow presented many challenges, but one of paramount importance was reliably cleaning up and concentrating PCR products in low elution volumes.
Optimization of the Mag-Bind® RxnPure Plus chemistry has yielded sequencable product in elution volumes as low as 15 µl. However, current automation methodology relies on standard magnetic separation products designed for reactions with a minimum elution volume 2X greater at 30 µl. This limitation is due to the way the PCR labware nests in the ring magnets resulting in the formation of a bead ring that is significantly elevated from the bottom of the well. Attempted elution volumes lower than 30 µl resulted in non-uniform mixing of elution buffer producing inconsistent product recovery and in some instances, nearly complete loss of elution product (Figure 1).
|Figure 1: Inconsistent product recovery using standard magnet plate. A. Agilent TapeStation 2200 chromatogram data peaks from sheared control sample (1) and purified products with proper mixing (2) and purified products with improper mixing (3 & 4). B. Wells visually observed in which magnetic beads did not re-suspend into solution after mixing on the Hamilton Microlab STAR.|
In order to increase the reliability and reproducibility of our high throughput workflow the Alpaqua Magnum FLX® Enhanced Universal Magnet Plate was inserted in place of our standard magnetic separator. The Magnum FLX addresses our primary problem as it is designed for elution volumes as low as 10 µl while simultaneously significantly decreasing separation time and allowing for an increased variation in acceptable labware. In order to accomodate the larger dimensions of the Magnum FLX, our Hamilton Microlab STAR was fitted with the SBS deep well carrier adaptor allowing the spring cushion technology of the Alpaqua plate to freely function and with adequate clearance for the pipette tips above labware.
|Figure 2: Highly consistent recovery using the Alpaqua Magnum FLX Magnet Plate. Measured concentration (ng/µl) of 32 cleaned-up and recovered PCR products. Average total DNA per sample clean-up = 362.6 ng. Average total DNA per sample post clean-up = 313.5 ng. Average total DNA recovery = 86.5%.|
|Figure 3: Statistical analysis of clean-up quantification.|
|0.66 ± 0.03||0.33 ± 0.02||1.96 ± 0.04||2.57 ± 0.11|
Table 1: Spectroscopic measurement of sample purity.
Efficiency of the Alpaqua Magnum FLX was tested in our PCR clean-up workflow with 32 replicates of a 25 µl PCR reaction in a 96-well PCR plate. The amplicon product was cleaned up using our standard automated Mag-Bind RxnPure Plus protocol adding 1.8 volumes of paramagnetic beads (45 µl) for initial binding. Two consecutive washes of 70% ethanol were followed by elution in 15 µl of 10 mM Tris buffer. Following elution, sample concentrations were measured using Promega's Quantus™ system and DNA purity was measured using the NanoDrop™ 2000.
Complete recovery was seen across all 32 samples with exceptional consistency and sufficient ng/µl concentrations to proceed to sequencing (Figure 2). Measured concentration was on average 20.93ng/µl with a standard deviation of 1.77 ng/µl (Figure 3).
Additionally, excellent sample purity results (Table 1) were achieved. Furthermore, the Alpaqua Magnum FLX plate was able to cut the time of the PCR clean-up magnetization time by 33%; from 12 minutes down to 8 minutes.
This testing demonstrated that method optimization needs to be extended to the technology of critical accessory components to acheive the most efficient and effective operations. The Alpaqua Magnum FLX plate and Omega Bio-tek Mag-Bind® RxnPure Plus provide a high throughput-capable option for laboratories desiring to automate their NGS workflows in which low elution volume DNA clean-ups are required.
|Omega Bio-tek||M1386-01||Mag-Bind® RxnPure Plus (50 mL)|
|Omega Bio-tek||M1386-02||Mag-Bind® RxnPure Plus (500 mL)|
|Alpaqua||A000400||Magnum FLX® Enhanced Universal Magnet Plate (96-well)|
|Omega Bioservices||OBS-16s-100||16s Sequencing on Illumina® MiSeq®|