An additional Python script (sequence_creation.py) was used to prepare the sequence table based on plate and well information. The sequence for each plate is built so that it is first analyzed in positive ion mode, followed by negative ionization. A filename is generated automatically and contains the date, a unique sample identifier (combination of library, plate number, and well location), the method used, and the polarity. The unique sample identifier is important because it is used for the automatic annotation and extraction of the acquired spectral data. Therefore, the unique sample identifier in the metadata column needs to be matched with a substring of the acquisition file name. Here, only this identifier is important in the name, enabling the addition of other prefixes and suffixes, for example, the date, polarity, or method. The script generates acquisition sequence files specific to the Xcalibur sequence layout using the flow injection-Orbitrap MS n method. This can be easily modified for other analysis platforms, depending on their layout.