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. Features:

• Modified Base Detection
  • Supports both DNA and direct RNA
    • RNA processing details
  • Three detection algorithms support broad range of applications
    • Alternative model (preferred)
    • Sample comparison
    • De novo
• Reference-anchored raw signal vizualization
• Raw signal analysis python API
• User-friendly model estimation methods with tutorial

References:

• H. H. Jeong, H. K. Yalamanchili, C. Guo C, J. M. Shulman, and Z. Liu De novo Identification of DNA Modifications Enabled by Genome-Guided Nanopore Signal Processing bioRxiv (2016). Journal

• tombo on GitHub
• tombo on Documentation

• Module Name: tombo (see the modules page for more information)
• tombo is multithreaded. Please match the number of threads to the number of allocated CPUs
• Example files in $TOMBO_TEST_DATA
• General reference data in /fdb/igenomes/

Allocate an interactive session and run the program. Sample session:

[user@biowulf]$ sinteractive --mem=5g --cpus-per-task=4 --gres=lscratch:10
salloc.exe: Pending job allocation 46116226
salloc.exe: job 46116226 queued and waiting for resources
salloc.exe: job 46116226 has been allocated resources
salloc.exe: Granted job allocation 46116226
salloc.exe: Waiting for resource configuration
salloc.exe: Nodes cn3144 are ready for job

[user@cn3144]$ cd /lscratch/$SLURM_JOB_ID
[user@cn3144]$ module load tombo
[user@cn3144]$ tar xvfz ${TOMBO_TEST_DATA}/${GZFILE} .
[user@cn3144]$ export REF="/fdb/igenomes/Saccharomyces_cerevisiae/UCSC/sacCer3/Sequence/WholeGenomeFasta/genome.fa"
[user@cn3144]$ tombo resquiggle $FAST5DIR $REF --processes $SLURM_CPUS_PER_TASK  --num-most-common-errors 5 --overwrite
[18:08:23] Loading minimap2 reference.
[18:08:24] Getting file list.
[18:08:24] Loading default canonical ***** DNA ***** model.
[18:08:24] Re-squiggling reads (raw signal to genomic sequence alignment).
5 most common unsuccessful read types (approx. %):
     2.7% (     93 reads) : Alignment not produced                                                          
     1.0% (     35 reads) : Poor raw to expected signal matching (revert with `tombo filter clear_filters`) 
     0.1% (      5 reads) : Read event to sequence alignment extends beyond bandwidth                       
     -----
     -----
100%|##################################################################| 3621/3621 [05:49<00:00, 10.36it/s]
[18:14:14] Final unsuccessful reads summary (3.7% reads unsuccessfully processed; 133 total reads):
     2.6% (     93 reads) : Alignment not produced                                                          
     1.0% (     35 reads) : Poor raw to expected signal matching (revert with `tombo filter clear_filters`) 
     0.1% (      5 reads) : Read event to sequence alignment extends beyond bandwidth                       
[18:14:14] Saving Tombo reads index to file.

[user@cn3144]$ tombo detect_modifications alternative_model --fast5-basedirs $FAST5DIR \
    --statistics-file-basename native.e_coli_sample \
    --alternate-bases dam dcm --processes $SLURM_CPUS_PER_TASK
    [18:20:03] Parsing Tombo index file(s).
    [18:20:03] Performing alternative model testing.
    [18:20:03] Performing specific alternate base(s) testing.
    [18:20:03] Calculating read coverage regions.
    [18:20:03] Calculating read coverage.
    [18:20:04] Performing modified base detection across genomic regions.
    100%|##############################################################| 2392/2392 [01:10<00:00, 33.89it/s]

[user@cn3144]$ tombo plot most_significant --fast5-basedirs $FAST5DIR \
    --statistics-filename native.e_coli_sample.dcm.tombo.stats \
    --plot-standard-model --plot-alternate-model dcm \
    --pdf-filename sample.most_significant_dcm_sites.pdf
    [18:23:05] Loading statistics from file.
    [18:23:06] Parsing Tombo index file(s).
    [18:23:06] Loading default canonical ***** DNA ***** model.
    [18:23:06] Preparing plot data.
    [18:23:08] Plotting.

[user@cn3144]$ tombo text_output browser_files --statistics-filename native.e_coli_sample.dam.tombo.stats \
    --file-types dampened_fraction --browser-file-basename native.e_coli_sample.dam
    [18:23:42] Loading statistics from file.
    [18:23:42] Parsing and outputting statistics wiggles.

[user@cn3144]$ tombo text_output browser_files --fast5-basedirs $FAST5DIR \
    --file-types coverage --browser-file-basename native.e_coli_sample
    [18:24:23] Parsing Tombo index file(s).
    [18:24:23] Getting and writing  coverage bedgraphs.
    [18:24:23] Calculating read coverage regions.
    [18:24:23] Calculating read coverage.   

Create a batch script file (e.g. tombo.sh). For example:

#!/bin/bash
cd /lscratch/$SLURM_JOB_ID
module load tombo
tar xvfz ${TOMBO_TEST_DATA}/${GZFILE} .
export REF="/fdb/igenomes/Saccharomyces_cerevisiae/UCSC/sacCer3/Sequence/WholeGenomeFasta/genome.fa"
tombo resquiggle $FAST5DIR $REF --processes $SLURM_CPUS_PER_TASK  --num-most-common-errors 5 --overwrite
....
....

Submit this job using the Slurm sbatch command.

sbatch --cpus-per-task=4 --mem=10g --gres=lscratch:100 tombo.sh