2KAPA Biosystems, Inc, Woburn, MA, USA
ChIP-Seq library preparation can be a challenging procedure to automate because of its low ChIP DNA input. This Application Note describes the successful automation of Illumina ChIP-Seq library preparation on the Eppendorf epMotion 5075 TMX, using the KAPA High-Throughput Library Preparation Kit from Kapa Biosystems. Size-selected libraries were prepared from as little as 1 ng of fragmented ChIP DNA. To obtain the recommended 100 ng of library material for sequencing on the Illumina Genome AnalyzerIIx, only 18 amplification cycles were needed for 1 ng of input DNA, or 9 cycles when starting library construction with 10 ng of ChIP DNA.
The Eppendorf epMotion is a multi-purpose liquid handling workstation that is suitable for many laboratory procedures. The epMotion 5075 TMX and other models in the family are ideal walk-away companions for labs that demand high efficiency, accuracy, and automated workflow. The system can be easily programmed to perform entire procedures such as nucleic acid extractions based on a wide selection of kits and reagents. Equipped with 12 positions on the worktable, it is capable of working with virtually any type of tube or plate. What's more, its built-in expandability allows integration of any labware of your choice on the fly. The on-deck mixer has an incorporated thermo unit, providing incubation capability needed for the NGS library preparation. Six dispensing tools covering a 1–1000 µL range are available in both single- and 8-channel formats to meet different throughput requirements. Additionally, a gripper can be added to transport plates around the worktable with ease.
We have previously demonstrated successful automation of a wide variety of common nucleic acid purification procedures from Promega, Macherey-Nagel, Invitrogen, and others. The rapid growth of Next Generation Sequencing (NGS) in recent years has created new applications for automated liquid handling, including library construction and quantification. To meet the growing demand for quality and throughput in this field, we have developed epMotion protocols that are compatible with library preparation chemistries utilized by major NGS platforms from Illumina, Life Technologies and Roche. Our most recent addition to this expanding application family involved automation of library construction using the KAPA High-Throughput Library Preparation Kit, and validation of this automated protocol for the construction of ChIP-Seq libraries.
NGS library construction workflows are comprised of repetitive liquid handling and incubation steps and are therefore ideal for automation. The epMotion 5075 TMX has been optimized to carry out a full library construction workflow—including end repair, A-tailing, adapter ligation, bead-based cleanups, dual-SPRI (Solid Phase Reversible Immobilization) size selection, and all on-deck incubations for 24 samples in less than 6 hours without the need for user interference. The same protocol can be quickly expanded to process up to 96 samples. The system is carefully calibrated to minimize the consumption of expensive reagents. Its high-precision pipetting tools[1,] further assure the production of high-quality libraries for downstream sequencing.
The successful implementation of a high-throughput NGS construction pipeline requires a reliable automation platform, combined with appropriate reagent formulation, packaging, and an optimized protocol. Many library preparation kits that are labeled “automation-friendly” are nevertheless poorly suited for automation, for reasons including: 1) impractical reaction setups, 2) suboptimal cleanup parameters, 3) unsuitable recommendations with respect to user-supplied consumables, particularly with regard to quality (affinity for nucleic acids [2,] and compatibility with the overall process (e.g. microtiter plates are incompatible with thermocycler incubation), and 4) the requirement for off-instrument size selection.
Kapa Biosystems (www.kapabiosystems.com) has recently upgraded its NGS Library Preparation Kit for Illumina sequencing to accommodate the specific requirements of automated liquid handling on the epMotion. Specifically, 1) reagent volumes have been tailored for processing 96 samples per kit (which facilitates inventory management), and generous excesses of all reagents are supplied to provide for the dead volumes required by automated liquid handling; 2) reaction setups have been optimized to avoid pipetting of viscous enzyme solutions and volumes less than 5 µL; 3) total reaction and cleanup volumes never exceed 200 µL to ensure compatibility with standard 96-well plates; 4) efficient, cost-effective, and automation-friendly reaction cleanups are achieved through implementation of the “with-bead” strategy developed at The Broad Institute of MIT & Harvard and Foundation Medicine[3,], and the kit includes the PEG/NaCl SPRI solution required for re-using beads. This “with-bead” strategy significantly reduces the consumption of beads and plasticware, and eliminates sample loss associated with the physical transfer of DNA solutions from one plate to the next. Combined with Eppendorf twin.tec semi-skirted PCR plates that exhibit very low affinity to nucleic acids, these improvements present an ideal solution for low-input library preparations such as ChIP-Seq. Because the automated “with-bead” protocol leads to a higher yield of adapter-ligated library fragments, fewer cycles of library amplification with the engineered KAPA HiFi HotStart DNA Polymerase are likely to be required, thereby further reducing the risk of bias [4, 5,] and other amplification artifacts. In addition, the automated protocol provides fully validated dual-SPRI size selection, which is performed on the epMotion without any user intervention.