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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 Bravo NGS workstation B in 96-well format. The 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 Bravo NGS workstation 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 Bravo NGS workstation are provided together with this protocol and a brief description of the steps performed by the liquid handling system, potentially providing a framework for setting the method up on liquid handling systems from other manufacturers. 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. 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.
Nagel et al. (Mon,) studied this question.