ont-tombo 1.4

Analysis of raw nanopore sequencing data.

Tombo Summary

Tombo is a suite of tools primarily for the identification of modified nucleotides from nanopore sequencing data.

Tombo also provides tools for the analysis and visualization of raw nanopore signal.

Installation

Basic tombo installation (python 2.7 and 3.4+ support)

# install via bioconda environment
conda install -c bioconda ont-tombo

# or install pip package (numpy install required before tombo for cython optimization)
pip install numpy
pip install ont-tombo[full]

Quick Start

Re-squiggle raw nanopore read files and call 5mC and 6mA sites.

Then, for 5mA calls, output genome browser wiggle format file and, for 6mA calls, plot raw signal around most significant locations.

# skip this step if FAST5 files already contain basecalls
tombo preprocess annotate_raw_with_fastqs --fast5-basedir path/to/fast5s/ \
    --fastq-filenames basecalls1.fastq basecalls2.fastq \
    --sequencing-summary-filenames seq_summary1.txt seq_summary2.txt \
    --processes 4

tombo resquiggle path/to/fast5s/ genome.fasta --processes 4 --num-most-common-errors 5
tombo detect_modifications alternative_model --fast5-basedirs path/to/fast5s/ \
    --statistics-file-basename sample.alt_modified_base_detection \
    --per-read-statistics-basename sample.alt_modified_base_detection \
    --alternate-bases 5mC 6mA --processes 4

# produces "estimated fraction of modified reads" genome browser files
# for 5mC testing
tombo text_output browser_files --statistics-filename sample.alt_modified_base_detection.5mC.tombo.stats \
    --file-types dampened_fraction --browser-file-basename sample.alt_modified_base_detection.5mC
# and 6mA testing (along with coverage bedgraphs)
tombo text_output browser_files --statistics-filename sample.alt_modified_base_detection.6mA.tombo.stats \
    --fast5-basedirs path/to/fast5s/  --file-types dampened_fraction coverage \
    --browser-file-basename sample.alt_modified_base_detection.6mA

# plot raw signal at most significant 6mA locations
tombo plot most_significant --fast5-basedirs path/to/fast5s/ \
    --statistics-filename sample.alt_modified_base_detection.6mA.tombo.stats \
    --plot-standard-model --plot-alternate-model 6mA \
    --pdf-filename sample.most_significant_6mA_sites.pdf

Detect any deviations from expected signal levels for canonical bases to investigate any type of modification.

tombo resquiggle path/to/fast5s/ genome.fasta --processes 4 --num-most-common-errors 5
tombo detect_modifications de_novo --fast5-basedirs path/to/fast5s/ \
    --statistics-file-basename sample.de_novo_modified_base_detection \
    --per-read-statistics-basename sample.de_novo_modified_base_detection \
    --processes 4

# produces "estimated fraction of modified reads" genome browser files from de novo testing
tombo text_output browser_files --statistics-filename sample.de_novo_modified_base_detection.tombo.stats \
    --browser-file-basename sample.de_novo_modified_base_detection --file-types dampened_fraction

RNA

All Tombo commands work for RNA data as well, but a transcriptome reference sequence must be provided for spliced transcripts.

The reasons for this decision and other tips for processing RNA data within the Tombo framework can be found in the RNA section of the detailed Tombo documentation.

Further Documentation

Run tombo -h to see all Tombo command groups and run tombo [command-group] -h to see all commands within each group.

Detailed documentation for all Tombo commands and algorithms can be found at https://nanoporetech.github.io/tombo/

Citation

Stoiber, M.H. et al. De novo Identification of DNA Modifications Enabled by Genome-Guided Nanopore Signal Processing. bioRxiv (2016).

http://biorxiv.org/content/early/2017/04/10/094672

Known Issues

• The Tombo conda environment (especially with python 2.7) may have installation issues.

  • Tombo works best in python 3.4+, so many problems can be solved by upgrading python.

  • If installed using conda:

    • Ensure the most recent version of conda is installed (conda update -n root conda).

    • It is recommended to set conda channels as described for bioconda.

    • Run conda update --all.

  • In python 2.7 there is an issue with the conda scipy.stats package. Down-grading to version 0.17 fixes this issue.

  • In python 2.7 there is an issue with the conda h5py package. Down-grading to version <=2.7.0 fixes this issue.