Our project is dedicated to analyzing sequence reads from a plasmid following its digestion by either a restriction enzyme or a Cas9/gRNA complex. The aim is the identification of cutting sites within the plasmid. The reads were generated by DNA sequencing with a MinION, a pocket-sized sequencing device based on nanopore technology. For the project, the FAST5 read files are provided. The binary FAST5 format standardizes nucleotide sequence data storage, ensuring compatibility across various bioinformatics tools. For the analysis, a suite of specialized tools is employed. The Guppy tool facilitates base calling and demultiplexing of nanopore sequence reads based on barcode identifiers. qfilter, a little tool developed in my lab, is utilized to filter sequencing data, retaining only high-quality reads while discarding noisy or low-quality data. The Minimap tool aligns long nucleotide sequences to a reference genome, facilitating the detection of similarities and differences relevant to cutting sites. This in turn is done with the Integrated Genome Viewer (IGV). Overall, the combined use of these tools enables the processing and analysis of MinION-derived sequencing data, ultimately leading to the identification of sequence features. Finally, the simulation of nanopore sequence reads is exemplified. All files from this chapter are available from the book’s GitHub repository at CompBiol3/21_NanoporeSeq .