Automated NGS Library Prep: Ligation Efficiency of NxSeq® Sample Preparation Using the Gilson PIPETMAX® 268

Introduction

As the number of samples for sequencing increases, there is a need for library prep automation that matches the throughput of the next generation sequencing (NGS) instruments while not requiring an investment equal to the sequencer itself. By utilizing the Gilson PIPETMAX^® 268, a novel NGS sample prep chemistry was automated to reduce the time to achieve the final prepared library, increasing the efficiency of the method and consistency of the resulting DNA library to be sequenced.

The NxSeq^®DNA sample prep chemistry combines typical end‐repair and A‐tailing steps into one master mix step with buffers directly compatible with downstream ligation steps, eliminating the need for multiple cleanup steps throughout the process. The chemistry has also been optimized to drive higher A‐tailing efficiencies, which reduces chimera formation from blunt fragments and loss of fragments tailed with nucleotides other than the necessary “A.” Chemistries designed by Lucigen and automated on the PIPETMAX allow numerous DNA libraries to be prepared simultaneously, including incorporation of barcoded adapters for multiplex PCR and sequencing. In contrast to other small, dedicated automation platforms, the PIPETMAX is open and programmable, meaning users can choose to utilize the platform for a range of other applications, including qPCR and cell‐based assays. In this application, NGS libraries were prepared from E. coli DH10B genomic DNA (gDNA) and FAM‐labeled adaptors using the NxSeq^® DNA Sample Prep kit, comparing the manual method with an automated library prep protocol on the PIPETMAX^®. This application note describes the results of the ligation efficiency of manual library preparation versus automated library preparation.

Materials & Methods

Materials

• E. Coli DH10B gDNA – Lucigen, Middleton, WI
• NxSeq DNA Sample Prep Kit – Lucigen, Middleton, WI
• FAM‐Labeled Adaptors – Integrated DNA Technologies, Coralville, IA
• Qubit 2.0 Fluorometer – Life Technologies, Grand Island, NY
• Qubit dsDNA HS Assay Kit – Life Technologies, Grand Island, NY
• Agencourt AMPure XP Beads – Beckman Coulter, Indianapolis, IN
• Biorad DNA Engine Thermocycler – Biorad, Hercules, CA
• Agilent Bioanalyzer 2100 and High Sensitivity DNA Kit – Agilent
  Technologies, Santa Clara, CA
• Synergy 2 Multi‐Mode Microplate Reader – Biotek, Winooski, VT
• PIPETMAX 268 with TRILUTION^® micro software – Gilson, Middleton, WI

Methods – Sample Preparation

1. E. coli DH10B gDNA was sheared by sonication to a size range of 50‐500 bp. The size was confirmed on the Agilent Bioanalyzer (Figure 1) and concentration readings were performed on the Qubit fluorometer.
2. End repair and A‐tailing were performed for manual libraries by preparing a master mix of NxSeq 2X End Repair Buffer, NxSeq End Repair Enzymes, and Low TE added to 500 ng of DNA per reaction. The samples were incubated on a thermocycler for 20 minutes at 25°C, 20 minutes at 72°C, and then held at 4°C.
3. For automated sample prep, end repair reagents, A‐tailing reagents, and gDNA were placed on the bed of the Gilson PIPETMAX® 268 according to the NxSeq® Sample Prep protocol preloaded on the PIPETMAX TRILUTION micro software, as shown by the layout in Figure 2. Once samples, master mix reagents, and enzymes were combined, the samples were removed from the bed and placed on a thermocycler for 20 minutes at 25°C, 20 minutes at 72°C, and then held at 4°C.
4. After incubation, FAM‐labeled adaptors and high concentration DNA ligase were added to the manually prepared libraries. Libraries prepared on the PIPETMAX were placed back onto the bed where the samples, FAM‐labeled adaptors, and high concentration ligase were combined using the PIPETMAX.
5. Both the manual and the PIPETMAX libraries were incubated at room temperature for 30 minutes.
6. During incubation, a stock of 1.7X sizing buffer was prepared by combining 30% PEG, 5M Sodium Chloride, and distilled water.
7. One replicate of both the manual and PIPETMAX libraries was size selected using 1.7X sizing buffer and AMPure XP beads. Another set of manual and PIPETMAX library replicates was size selected using a two‐step bead cleanup. DNA was size selected with a 0.8X ratio of beads to DNA (ratio based on volume) for large fragment removal, followed by a 0.2X ratio of beads to DNA for small fragment removal. After size selection, 1 μL of each library and 1 μL of diluted 1:10 sheared gDNA were run on a High Sensitivity DNA Chip on the Agilent Bioanalyzer.

>> Download the full Application Note as PDF

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