Hamilton STAR: automated single-stranded DNA library preparation (ssDNA2.0) v1

We here provide an implementation of the ssDNA2.0 single-stranded library preparation method (Gansauge et al. 2017, Gansauge et al. 2020) for automated liquid handling on the Hamilton NGS STAR workstation in 96-well format. The protocol derives from a previous implementation of the ssDNA2.0 method on the Bravo workstation (Nagel et al. 2024). The ssDNA2.0 method was developed primarily for ancient DNA research but is also suitable for library preparation from other sources of degraded or single-stranded DNA, such as cell-free DNA, formalin-fixed samples or synthetic oligonucleotides. To use the protocol, a Hamilton NGS STAR workstation is required. For a fully walk-away solution that does not require adding pipette tips during the run, the addition of a Cytomat (Thermo Fisher Scientific) 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 Hamilton STAR workstation are provided together with this protocol and a brief description of the steps performed by the liquid handling system. 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 manual ssDNA 2.0 library preparation, please refer to Gansauge et al. 2020. Note that the adapter sequences added with this method differ from standard Illumina adapters by a 5-bp deletion in the P5-adapter, requiring a custom sequencing primer (see Gansauge et al. 2020). This protocol consists of two parts: (i) library preparation, with or without Uracil-DNA-glycosylase treatment for the removal of internal uracils from ancient DNA molecules, and (ii) library quantification by quantitative real-time PCR. A protocol for library amplification and indexing is provided separately. Two control oligonucleotides are added during library preparation. CL304 (60 million molecules) is added at the beginning of the workflow to assess the efficiency of DNA conversion into sequencing libraries (Glocke & Meyer 2017) and to identify inhibition or saturation in individual reactions. TL250 (1 million molecules) is added during the elution step for additional quality control. References Gansauge, M.-T., Gerber, T., Glocke, I., Korlevic, P., Lippik, L., Nagel, S., Riehl, L. M., Schmidt, A., & Meyer, M. (2017). Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase. Nucleic Acids Research, 45(10): e79. Gansauge, M.-T., Aximu-Petri, A., Nagel, S., & Meyer, M. (2020). Manual and automated preparation of single-stranded DNA libraries for the sequencing of DNA from ancient biological remains and other sources of highly degraded DNA. Nature Protocols, 15, 2279-2300. Glocke, I., & Meyer, M. (2017). Extending the spectrum of DNA sequences retrieved from ancient bones and teeth. Genome Research, 27, 1230-1237. Nagel, S., Schmidt, A., Aximu-Petri, A., Patova, A., Szymanski, M., Essel, E. & Meyer, M. (2026). Bravo workstation: automated single-stranded DNA library preparation (ssDNA2.0). protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg32bdpv25/v2