Hamilton NGS Star workstation: automated in-solution hybridization capture (MPI-EVA method) v1
Authors: Nagel Sarah , Schmidt Anna , Nickel Birgit , Zorn Julia , Szymanski Merlin , Essel Elena , Meyer Matthias 
Published: January 2025

We here provide a protocol for automated hybridization capture of ancient DNA libraries on the Hamilton NGS Star in 96-well format using a capture method originally developed by Gnirke et al. 2009 and modified at the MPI-EVA for use with ancient DNA by Fu et al. 2013. Modifications to the latter method include the omission of a wash with Wash Buffer 3 and the hydroxide melt step for the elution of the captured library molecules prior to amplification. In-solution hybridization capture is performed using single-stranded biotinylated DNA probes, which can be generated from oligonucleotide arrays, synthetic oligonucleotide pools or other sources. It has recently been shown that small amounts of cross-contamination can occur in hybridization capture as a result of 'PCR jumping' during post-capture amplification even when working with double-indexed libraries (Zavala et al. 2022). The choice of amplification conditions described here (polymerase, primers and thermal profile) minimizes PCR jumping, despite amplifying libraries into PCR plateau. Yet, we strongly urge users to quantify cross-contamination in the resulting sequence data using the computational strategy described by Zavala et al. 2022. To use the protocol, a Hamilton NGS Star system with a custom deck layout is required. Calibration of the instrument for this protocol has to be performed by the user and requires significant expertise in using the platform. Electronic protocol files for the NGS Star are provided together with this protocol and a brief description of the steps performed by the liquid handling system. Documents detailing the preparation of the probes and other reagents are also provided. Some of the instructions, for example regarding the documentation and location of files, are specific to the environment and workflows of the Ancient DNA Core Unit of the MPI-EVA and have to be amended in other environments. We strongly advise against manual execution of the protocol, as manual handling lacks the precision of automated liquid handling and may lead to cross-contamination of samples or non-optimal results. For a manual protocol, please refer to Zavala et al. 2022. This protocol consists of several parts: (i) preparation of the sample plate, (ii) hybridization over-night, (iii) post-capture washing, library amplification and clean-up, and (iv) qPCR quantification of capture yields. Capture can be performed in a single or two successive rounds of hybridization capture, with two-round capture being our current default. References Fu, Q., Meyer, M., Gao, X., Stenzel, U., Burbano, H. A., Kelso, J., & Pääbo, S. (2013). DNA analysis of an early modern human from Tianyuan Cave, China. Proceedings of the National Academy of Sciences of the United States of America, 110, 2223-2227. Gnirke, A., Melnikov, A., Maguire, J., Rogov, P., LeProust, E.M., Brockman, W., Fennell, T., Giannoukos, G., Fisher, S., Russ, C., Gabriel, S., Jaffe, D. B., Lander, E. S., Nusbaum, C. (2009) Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat Biotechnol. 27(2), 182-189 Zavala, E. I., Ayinuer-Petri, A., Richter, J., Nickel, B., Vernot, B., & Meyer, M. (2022). Quantifying and reducing cross‐contamination in single‐ and multiplex hybridization capture of ancient DNA. Molecular Ecology Resources, 22(6), 2196-2207.