Text Outputs

Tombo provides two text outputs:

  1. Genome Broser Files - Genome browser compatible per-genomic-base statistics

  2. Fasta - Genomic sequence output surrounding identified modified base sites

tombo text_output browser_files

The tombo text_output browser_files command takes in a set of reads (--fast5-basedirs) and/or a statistics file generated from a tombo detect_modifications command (--statistics-filename). A control set of reads can also be provided (--control-fast5-basedirs). Output files will be produced for each requested statistic (both plus and minus strands) in variableStep wiggle format (or bedgraph format for --file-type coverage).

Several statistics are available for output:

  • coverage - The coverage level for mapped and validly re-squiggled reads

  • valid_coverage - The coverage level for reads that are mapped, validly re-squiggled and outside the interval specified by --single-read-threshold specified in a --statistics-filename.

  • dampened_fraction - The estimated fraction of significantly modified reads (non-level_sample_compare modified base detection methods only)

  • fraction - The raw fraction of significantly modified reads (non-level_sample_compare modified base detection methods only)

  • statistic - Statistic produced from level_sample_compare method (default outputs the effect size statistic; if --store-p-value is specified to detect_modfications command then the negative log p-value is output).

  • signal - The mean signal level across all reads mapped to this location

  • signal_sd - The mean signal standard deviation across all reads mapped to this location (not available unless --include-event-stdev was provided in tombo resquiggle command)

  • dwell - The mean number of raw observations observed assigned to this location

  • difference - The difference in normalized signal level between a sample and control set of reads


The dampened_fraction output adds psuedo-counts to the detected number of un-modified and modified reads at each tested location (as specified by the --coverage-dampen-counts option), while the fraction option returns the raw fraction of modified reads at any reference site from detect_modifications results. The dampen_fraction output is intended to allow the inclusion of low coverage regions in downstream analysis without causing potentially false positive site at the top of rank lists. Visualize different values of the --coverage-dampen-counts option with the included scripts/test_beta_priors.R script.

Motif Filtering Output

The tombo text_output browser_files contains options --motif-descriptions and --genome-fasta enabling computed statistics output to be restrcited to only those locations at known/putative motif-centered modifications. These options apply to the fraction, dampened_fraction and valid_coverage file types.


signal, signal_sd, dwell and difference require each reads’ event level data to be extracted from the raw read files and thus may be quite slow. valid_coverage, fraction , dampened_fraction and statistic can be extracted from the tombo statistics files and coverage from the Tombo index, which is much faster.

The signal, signal_sd, dwell and difference outputs all require the --fast5-basedirs option, the valid_coverage, fraction , dampened_fraction and statistic outputs require the --statistics-filename option, and coverage output requires one or the other.

Files will be output to individual wiggle files (two per statistic for plus and minus genomic strand) in the following format [wiggle-basename].[wiggle-type].[sample|control]?.[plus|minus].wig

tombo text_output signif_sequence_context

The tombo text_output signif_sequence_context command writes the genome sequence surrounding unique genomic positions with the largest estimated fraction of modified bases. This can be useful for several tasks related to modified base detection including motif discovery.

To run tombo text_output signif_sequence_context, a --statistics-filename is required to extract the most significant locations and either a --fast5-basedirs or --genome-fasta is required to extract the genomic sequence. Several options are availble for selecting the sequence to be output:

  • --num-regions - Defines the number of unique locations to be output

  • --num-bases - Defines the number of bases to be output surrounding the significant locations

The output of this command could be used to determine sequence contexts consistently modified within a sample. Example meme command line modified base motif detection command.

tombo detect_modifications de_novo --fast5-basedirs <fast5s-base-directory> \
             --statistics-file-basename sample.de_novo
tombo text_output signif_sequence_context --statistics-filename sample.de_novo.tombo.stats \
             --genome-fasta reference.fasta --num-regions 1000 --num-bases 50
./meme -oc tombo.de_novo_motif_detection.meme -dna -mod zoops tombo_results.significant_regions.fasta