This repository contains the electronic protocol files for 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.
It provides the following 5 electronic protocols:
- library preparation, with or without Uracil-DNA-glycosylase treatment
- library quantification by quantitative real-time PCR.
- Preparation of index-primer aliquots for 96 or 384 well plates
- library indexing
- library purification
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.
A brief description of the steps performed by the liquid handling system is provided for each protocol in the docs/ directory, 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.
More information is provided in the Supplementary Material published in Gansauge et al. 2020.
The electronic files are also available on Zenodo
Gansauge, Marie-Theres, Tobias Gerber, Isabelle Glocke, et al., Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase, Nucleic Acids Research, Volume 45, (2017). https://doi.org/10.1093/nar/gkx033
Gansauge, Marie-Theres, Ayinuer Aximu-Petri, Sarah Nagel et al. 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, Volume 15, (2020). https://doi.org/10.1038/s41596-020-0338-0