ont-tombo 1.0

Perform re-squiggle algorithm (once per root directory of FAST5 files)

* Need not contain Events data, but must contain Fastq slot

tombo resquiggle path/to/amplified/dna/fast5s/ genome.fasta --minimap2-executable ./minimap2 --processes 4

Identify modified nucleotides comparing to an alternative 5mC model

tombo test_significance --fast5-basedirs path/to/native/dna/fast5s/ --alternate-bases 5mC --statistics-file-basename sample_compare
tombo write_wiggles --fast5-basedirs path/to/native/dna/fast5s/ --wiggle-basename sample_compare.5mC --statistics-filename sample_compare.5mC.tombo.stats

Identify modified nucleotides from a single sample

tombo test_significance --fast5-basedirs path/to/native/dna/fast5s/ --statistics-file-basename sample --processes 4
tombo write_wiggles --fast5-basedirs path/to/native/dna/fast5s/ --wiggle-basename sample --statistics-filename sample.tombo.stats

Identify modified nucleotides comparing two samples

tombo test_significance --fast5-basedirs path/to/native/dna/fast5s/ --control-fast5-basedirs path/to/amplified/dna/fast5s/  --statistics-file-basename sample_compare
tombo write_wiggles --fast5-basedirs path/to/native/dna/fast5s/ --control-fast5-basedirs path/to/amplified/dna/fast5s/ --wiggle-basename sample_compare --statistics-filename sample_compare.tombo.stats

Identify modified RNA nucleotides from a single sample

tombo resquiggle path/to/native/rna/fast5s/ transcriptome.fasta --minimap2-executable ./minimap2 --processes 4
tombo test_significance --fast5-basedirs path/to/native/rna/fast5s/ --statistics-file-basename rna_sample --processes 4
tombo write_wiggles --fast5-basedirs path/to/native/rna/fast5s/ --wiggle-basename rna_sample --statistics-filename rna_sample.tombo.stats

Some plotting examples

tombo plot_max_coverage --fast5-basedirs path/to/native/rna/fast5s/ --tombo-model-filename /path/to/tombo/root/tombo/tombo_models/tombo.DNA.model
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/ --deepest-coverage
tombo plot_per_read --fast5-basedirs path/to/native/rna/fast5s/ --genome-locations chromosome:1000 chromosome:2000:- --alternate-model-filename /path/to/tombo/root/tombo/tombo_models/tombo.DNA.5mC.model

Extract sequences surrounding modified positions

tombo write_most_significant_fasta --statistics-filename sample_compare.5mC.tombo.stats --genome-fasta genome.fasta

Resquiggle (Must be run before any other commands):

resquiggle                    Re-annotate raw signal with genomic aignement of existing basecalls.

Statistical Testing Command:

test_significance             Test for shifts in signal against a reference or against another set of reads.

Text Output Commands:

write_wiggles                 Write wiggle files for nanopore signal values, coverage, and statistics.
write_most_significant_fasta  Write sequence where signal differs the most significantly between two groups.

Genome Anchored Plotting Commands:

plot_max_coverage             Plot signal in regions with maximal coverage.
plot_genome_location          Plot signal at defined genomic locations.
plot_motif_centered           Plot locations centered on a specific motif.
plot_max_difference           Plot locations where signal differs the most between two groups.
plot_most_significant         Plot locations where signal differs most significantly between two groups.
plot_model_most_significant   Plot locations where signal differs most significantly from the kmer model.
plot_motif_with_stats         Plot signal from several regions and test statistics centered on a k-mer of interst.
plot_per_read                 Plot per read modified base predictions.

Sequencing Time Anchored Plotting Commands:

plot_correction               Plot segmentation before and after correction.
plot_multi_correction         Plot multiple raw signals anchored by genomic location.

Other Plotting Commands:

cluster_most_significant      Clustering traces at bases with significant differences.
plot_kmer                     Plot signal quantiles acorss kmers.

Read Filtering (Only effects tombo index file):

clear_filters                 Clear filters to process all successfully re-squiggled reads.
filter_stuck                  Apply filter based on observations per base thresholds.

Event-based Re-squiggle (Primarily for producing new models):

event_resquiggle              Re-annotate raw signal with genomic alignment from existing basecalls.
model_resquiggle              Re-annotate raw signal with genomic bases by shifting the signal to more closely match a k-mer model.
estimate_kmer_reference       Estimate reference k-mer model derived from the provided reads.
estimate_alt_reference        Estimate alternative tombo model from a sample containing standard bases spiked with a single non-standard base at random positions.

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

At least one supported mapper:

• minimap2 (https://github.com/lh3/minimap2)

• BWA-MEM (http://bio-bwa.sourceforge.net/)

• graphmap (https://github.com/isovic/graphmap)

• HDF5 (http://micro.stanford.edu/wiki/Install_HDF5#Install)

python Requirements (handled by pip):

• numpy (must be installed before installing tombo)

• scipy

• h5py

• cython

Optional packages for plotting (install R packages with install.packages([package_name]) from an R prompt):

• rpy2 (along with an R installation)

• ggplot2 (required for any plotting subcommands)

• cowplot (required for plot_motif_with_stats subcommand)

Optional packages for alternative model estimation:

• sklearn

pip install ont-tombo[full]

pip install ont-tombo[plot]

pip install ont-tombo[alt_est]

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