The document describes a simple and flexible automated library purification solution using Caliper's Zephyr Genomics Workstation. The system utilizes solid-phase paramagnetic bead technology to purify DNA fragments over 100 bp in under an hour. It processes between 8 to 96 samples using preinstalled methods without needing programming. This allows users to prepare varying molecular biology steps for next-generation sequencing library preparation while samples are purified. The system improves purification consistency and conserves reagents without sacrificing efficiency or recovery.

1. A Simple and Flexible Solution for Automated Library Purification
Isaac Meek2, Hui Xu1, Elaine Wong-Ho1 and Josh Molho1
1
Caliper Life Sciences, Alameda, CA, 2Caliper Life Sciences, Hopkinton, MA
Abstract Method and Materials Results and Discussion
As the throughput of second generation sequencers continues to increase, so does the need for Method Run time for SPRI method is <1 hour, which allows the user to focus on setting up enzymatic reactions
automation solutions that ease the tedious library construction process. Unlike traditional Sanger-based or other intensive activities. Modifications to the method can be implemented by the user and saved as
The Agencourt AMPure Purification system utilizes solid-phase
sequencing protocols, second generation sequencing chemistries can very greatly from application to a separate “modified” method. Modifications include extended drying, increased tip mixing, as well as
paramagnetic bead technology for high-throughput purification
application, making a full automation solution challenging to implement for most laboratories. In volume changes for the washes and elution.
of DNA fragments. Agencourt AMPure utilizes an optimized
response to the growing need for automation and standardization, Caliper has developed a solution that
buffer to selectively bind PCR amplicons 100 bp and larger to
efficiently handles the routine purification part of NGS workflows, with no programming or optimization
paramagnetic beads. Excess oligos, nucleotides, salts, and
needed. Utilizing preinstalled methods, a simplified graphical user interface (GUI), software safe
enzymes can be removed using a simple washing procedure.
guards, and the commonly used SPRI chemistry, the Zephyr Genomics Workstation allows true walk
The resulting sample is essentially free of contaminants and is
away automation, allowing the user to prepare the varying molecular biology steps in the library
commonly used as the primary purification method between Figure 4. Typical recovery after sizing on Caliper’s
production process while samples are being purified. LabChip XT instrument. The sample was then
enzymatic steps in next gen sequencing library preparation. processed via the standard Illumina paired-end protocol
Leveraging the partial tip loading capability of the instrument it can process between 8 and 96 samples with SPRI purifications between each subsequent steps
in approximately 45 minutes. The integrated ultrasonic sensor allows for the noncontact detection of (see figure 1).
reagents and consumables, ensuring correct deck layout prior to starting the method. In addition to
improved purification consistency, Caliper’s approach conserves reagent without sacrificing efficiency or
recovery. The presented poster will describe the performance of the chemistry on the liquid hander, as
well as the operation and setup of the instrument.
1. Select method and
enter contact SMS
Figure 5. Electropherogram of an adapter-ligated
Introduction text Email address paired-end library that was sized using Caliper’s LabChip
XT fractionation instrument and purified using
2. Choose number of magnetic-bead based purification. Note: the change in
The Zephyr Genomics Workstation from Caliper Life Sciences is a compact liquid handler designed to samples to process, size from the fragmented sample is due to the addition of
automate routine molecular biology processes without the need for programming expertise. The 8 to 96 samples sequencing adapters, and not a result of the purification
process.
Workstation hardware includes the compact Zephyr liquid handler equipped with a 96-well high volume 3. Ensure deck is
head (HVH) with disposable tips, consumables gripper, a vacuum filtration station, and a unique populated correctly.
ultrasonic, non-contact liquid level sensor. Deck map provides
A robust and easy to use GUI allows for the operator to select from a library of pre-developed methods consumables and 4. Review and start
and provides a guide for assay setup. A Deck Map is provided to give the operator a graphical view of reagent volumes method.
the consumables and reagents needed and where they belong. With only a few clicks, the operator can Figure 2. outlines the method selection and deck setup workflow for the Zephyr Genomics Workstation. 1) Select method and enter contact information;
setup a method and allow the method to run to completion unattended. SMS text or email. 2) Choose number of samples to process, 8 to 96 in multiples of 8. 3) Ensure that deck is populated correctly. The deck map provides Figure 6. LabChip GX electropherogram of PCR
consumable information and reagent volumes. 4) Review method and select “start”. Method runs to completion and will notify user defined in step 1 when products purified on the Zephyr Genomics Workstation.
method is comlplete. The GX can be utilized for routine high throughput sizing
of sheared genomic DNA, total RNA, and purified
libraries.
Figure 3. Outlines the steps outlined
in the Agencourt AMPure purification
protocol. The chemistry is magnetic
bead-based and is easily scalable to
meet the throughput of the user.
Fragment Size Yield CV Max Min
Table 1. Recovery values for PCR fragments (BP) (%) (%) (%) (%)
Illustration by Agencourt purified using the Zephyr and analyzed on
the GX using the DNA 5k assay. 1008 89.0 5.2 98.8 82.4 n=16
Materials 555 90.5 4.6 89.4 82.4 n=16
Consumables
96 well Elution Plate, Costar 3650
200 μL Tips, Caliper Life Science
Conclusion
12 Channel Reservoir, Seahorse #S30028
Reagents The Zephyr Genomics Workstation can be utilized for the time consuming purifications between the
enzymatic steps in library construction. Up to 96 samples can be processed in <1 hour with a high
Agencourt AMPure SPRI magnetic beads recovery rate. The system can also be used for filtration based chemistries, such as the Qiagen QiaQuick
96, and enzymatic reaction setup. The operation is simple, fast and fully automated on the Zephyr
Fresh 70% EtOH
Genomics Workstation.
10 mM TRIS-Acetate, pH 8.0 for elution
Hardware
References
Agencourt SPRIPlate magnetic plate
1. Sample Preparation for Genomic DNA Paired End Libraries, www.illumina.com
Illustration by Illumina
Caliper Zephyr Genomics Workstation 2. AMPure PCR purification, www.agencourt.com
Figure 1. Outlines the Illumina paired-end workflow, which includes numerous enzymatic steps that require purification after
each. Due to the number of enzymatic steps, high recovery and reproducibility is key to retaining mass and sample integrity.