Scripts for analysing SMF data from Krebs Lab @ EMBL. These scripts depend on functions (included) for calling methylation and single molecule TF analysis. Exemplary input files and objects are provided for all analyses.
When starting from raw sequencing reads, use an input file to point at reads (exemplified), run SMF_align script and align reads via QuasR. When done (or starting directly from alignments), use an input file (exemplified) for QuasR to point at alignments. Run context_methylation_call to call and analyse methylation genome-wide with context information. Use sort_TF_states and sort_TF_states_clusters to generate single molecule methylation vectors based on your analysis type. We recommend starting with single motifs and using sort TF_states. Run plot_single_molecule_examples script to plot the data, including average methylation and single molecule stacks.
Please set your working directory as specified in the script, or use our downloaded github folder. If you create 2 subdirectories in our downloaded GitHub folder, titled “rds” and “tmp”, the scripts will generate the output in these folders.
We provide an exemplary input file for alignment via QuasR. (QuasR_aln_input) We provide an exemplary input file for analysing BAMs via QuasR. (QuasR_input)
We provide an exemplary R objects for TF motif lists, needed in sort_TF_states. (mapped_jaspar_ChIP_bound_motifs.rds) This object is also used to generate an object for TF clusters, needed in sort_TF_states_clusters.
We provide an exemplary R object for amplicon coordinates, which is needed if analysing PCR-based data. This can be used as an example, if you are interested in plotting information for your regions of interest.