ont-tombo 1.2

Re-squiggle (Raw Data Alignment)

tombo resquiggle path/to/amplified/dna/fast5s/ genome.fasta --processes 4

Only R9.4/5 data is supported at this time.

DNA or RNA is automatically determined from FAST5s (set explicitly with --dna or --rna).

FAST5 files need not contain Events data, but must contain Fastq slot. See annotate_raw_with_fastqs for pre-processing of raw FAST5s.

Identify Modified Bases

# comparing to an alternative 5mC and 6mA model (recommended method)
tombo test_significance --fast5-basedirs path/to/native/dna/fast5s/ \
    --alternate-bases 5mC 6mA --statistics-file-basename sample

# comparing to a control sample (e.g. PCR)
tombo test_significance --fast5-basedirs path/to/native/dna/fast5s/ \
    --control-fast5-basedirs path/to/amplified/dna/fast5s/ --statistics-file-basename sample_compare

# compare to the canonical base model
tombo test_significance --fast5-basedirs path/to/native/dna/fast5s/ \
    --statistics-file-basename sample_de_novo --processes 4

Must run resquiggle on reads before testing for modified bases.

test_significance produces a binary file. See write_wiggles for several text outputs or plot_most_significant to use for genome region selection.

Text Output (Wiggle file format)

# extract fraction of reads modified at each genomic base in wiggle file format
tombo write_wiggles --wiggle-types fraction --statistics-filename sample.5mC.tombo.stats

# extract read depth from mapped and re-squiggled reads
tombo write_wiggles --wiggle-types coverage --fast5-basedirs path/to/native/dna/fast5s/

Extract Sequences Surrounding Modified Positions

tombo write_most_significant_fasta --statistics-filename sample.6mA.tombo.stats \
    --genome-fasta genome.fasta

Plotting Examples

# plot raw signal with standard model overlay at reions with maximal coverage
tombo plot_max_coverage --fast5-basedirs path/to/native/rna/fast5s/ --plot-standard-model

# plot raw signal along with signal from a control (PCR) sample at locations with the AWC motif
tombo plot_motif_centered --fast5-basedirs path/to/native/rna/fast5s/ \
    --motif AWC --genome-fasta genome.fasta --control-fast5-basedirs path/to/amplified/dna/fast5s/

# plot raw signal at genome locations with the most significantly/consistently modified bases
tombo plot_most_significant --fast5-basedirs path/to/native/rna/fast5s/ \
    --statistics-filename sample.5mC.tombo.stats --plot-alternate-model 5mC

# plot per-read test statistics using the 6mA alternative model testing method
tombo plot_per_read --fast5-basedirs path/to/native/rna/fast5s/ \
    --genome-locations chromosome:1000 chromosome:2000:- --plot-alternate-model 6mA

Re-squiggle (Raw Data Alignment):

Must be run before any other commands.

resquiggle                    Re-annotate raw signal with genomic alignment from existing basecalls.

Modified Base Detection:

test_significance             Test for shifts in signal indicative of non-canonical bases.

Text Output Commands:

write_wiggles                 Write text outputs for genome browser visualization and bioinformatic processing (wiggle file format).
write_most_significant_fasta  Write sequence centered on most modified genomic locations.

Genome Anchored Plotting Commands:

plot_max_coverage             Plot raw signal in regions with maximal coverage.
plot_genome_location          Plot raw signal at defined genomic locations.
plot_motif_centered           Plot raw signal at a specific motif.
plot_max_difference           Plot raw signal where signal differs most between two read groups.
plot_most_significant         Plot raw signal at most modified locations.
plot_motif_with_stats         Plot example signal and statistic distributions around a motif of interst.
plot_per_read                 Plot per read modified base probabilities.

Read Filtering:

clear_filters                 Clear filters to process all successfully re-squiggled reads.
filter_stuck                  Apply filter based on observations per base thresholds.
filter_coverage               Apply filter to downsample for more even coverage.

Get additional help for subcommands with tombo [command] -h

python Requirements (handled by conda or pip):

• numpy

• scipy

• h5py

• cython

• mappy

Optional packages (handled by conda, but not pip):

• Plotting Packages

  • R

  • rpy2

  • ggplot2

  • gridExtra (required for plot_motif_with_stats and plot_kmer subcommands)

• On-disk Random Fasta Access

  • pyfaidx

pip install ont-tombo

pip install git+https://github.com/nanoporetech/tombo.git