Automated DNA Cleanup for NGS Workflow
Introduction
Next-generation sequencing (NGS) is an ever-increasingly utilized application in a myriad of fields ranging from basic biological research to pharmacogenetics and clinical medicine. An integral part of the initial steps of a typical NGS workflow involves multiple DNA cleanup steps. To better streamline NGS workflows, a need for advancements in higher throughput processing and turnaround time is crucial. To address this, Omega Bio-tek has developed a fully automatable protocol using Mag-Bind® TotalPure NGS beads (M1378) which can be employed in cobination with a liquid handler to perfor integral cleanup and size selection steps for library preparation. The capabilities of this method for high throughput cleanup were analyzed by DNA recovery and quality. Here, Omega Bio-tek’s Mag-Bind® TotalPure NGS beads are capable of efficiently cleaning up 96 samples in less than 35 minutes on a liquid handler, thereby greatly optimizing throughput and processing time for a more efficient implementation in NGS workflows.
Methods
Illustrated Protocol for Mag-Bind® TotalPure NGS automated cleanup
Figure 1. The above protocol can be implemented with a liquid handler and Omega Bio-tek’s Mag-Bind® TotalPure NGS Kit (M1378) for automated cleanup and library preparation in NGS workflow.
Results
Mag-Bind® TotalPure NGS from Omega Bio-tek performs excellent double-sided size selection
Mag-Bind® TotalPure NGS performs excellent RNA cleanup in a wide concentration range
Figure 1. Electropherogram overlay of the double-sided size selection on sheared gDNA at 0.8x/0.7x ratio set using Omega Bio-tek’s Mag-Bind® TotalPure NGS and a comparable kit from Company A following Manufacturer’s recommended protocols. The DNA was eluted in 25 µL and analyzed on Agilent’s TapeStation® 2200.
Figure 2. 10 µL of RNA at 50 ng/µL was cleaned up with Omega Bio-tek’s Mag-Bind® TotalPure NGS following manufacturer’s recommended protocols. The RNA was eluted in 20 µL and analyzed on Agilent’s TapeStation® 2200. Recovery rates were 85-92%, respectively.
Mag-Bind® TotalPure NGS has excellent recovery of targeted DNA fragments
Mag-Bind® TotalPure NGS performs excellent double-sided size selection on DNA from NGS library prep
Figure 3. 10 µL of 50 bp ladder was purified with Omega Bio-tek’s Mag-Bind® TotalPure NGS and a comparable product from Company A following manufacturer’s recommended protocols. The DNA was eluted in 20 µL and analyzed on Agilent’s TapeStation® 2200.
Figure 4. Next-generation sequencing libraries prepared from 350 ng sheared genomic DNA using Kapa Biosystem’s Hyper-Prep Kits (KK8504) and Omega Bio-tek’s Mag-Bind® TotalPure NGS and a comparable productfrom Company A on the Hamilton Microlab® STAR™. Mag-Bind® TotalPure NGS was used for 2 clean up step (0.8x and 1.0x) following Kapa Biosystem’s recommended protocol for cleanup. DNA was analyzed on Agilent’s TapeStation® 2200 following library construction.
Conclusions
Omega Bio-tek’s Mag-Bind® TotalPure NGS Kit (M1378) presents a myriad of advantages:
- High Throughput Capabilities
- Automated NGS library cleanup
- Excellent size selection
WP-0049
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Mag-Bind® TotalPure NGS
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