Poretools is a toolkit for manipulating and exploring nanopore sequencing data sets. Poretools operates on individual FAST5 files, directory of FAST5 files, and tar archives of FAST5 files.

References:

• N. Loman and A. Quinlan. Poretools: a toolkit for analyzing nanopore sequence data. bioRxiv. Also published in Bioinformatics 2014, 30:3399-3401. PubMed |  PMC |  Journal

• Manual
• GitHub

• Module Name: poretools (see the modules page for more information)
• Example files in $PORETOOLS_TEST_DATA

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

[user@biowulf]$ sinteractive --mem=6g
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]$ module load poretools
[user@cn3144]$ poretools --help
usage: poretools [-h] [-v]
                 {combine,fastq,fasta,...,yield_plot,occupancy,organise}
                 ...

optional arguments:
  -h, --help            show this help message and exit
  -v, --version         Installed poretools version

[sub-commands]:
  {combine,fastq,fasta,...,yield_plot,occupancy,organise}
    combine             Combine a set of FAST5 files in a TAR achive
    fastq               Extract FASTQ sequences from a set of FAST5 files
    fasta               Extract FASTA sequences from a set of FAST5 files
    stats               Get read size stats for a set of FAST5 files
    hist                Plot read size histogram for a set of FAST5 files
    events              Extract each nanopore event for each read.
    readstats           Extract signal information for each read over time.
    tabular             Extract the lengths and name/seq/quals from a set of
                        FAST5 files in TAB delimited format
    nucdist             Get the nucl. composition of a set of FAST5 files
    metadata            Return run metadata such as ASIC ID and temperature
                        from a set of FAST5 files
    index               Tabulate all file location info and metadata such as
                        ASIC ID and temperature from a set of FAST5 files
    qualdist            Get the qual score composition of a set of FAST5 files
    qualpos             Get the qual score distribution over positions in
                        reads
    winner              Get the longest read from a set of FAST5 files
    squiggle            Plot the observed signals for FAST5 reads.
    times               Return the start times from a set of FAST5 files in
                        tabular format
    yield_plot          Plot the yield over time for a set of FAST5 files
    occupancy           Inspect pore activity over time for a set of FAST5
                        files
    organise            Move FAST5 files into a useful folder hierarchy


[user@cn3144]$ cp ${PORETOOLS_TEST_DATA}/ERA484348_014370_subset.tar .
[user@cn3144]$ ls -lh ERA484348_014370_subset.tar
-rw-r--r-- 1 user group 2.1G Jun 30 08:42 ERA484348_014370_subset.tar

Extract fastq format sequences from all the pass reads in the example data set. Note that poretools can work on a tar archive directly - no need to extract the archive and create large numbers of small files that can degrade file system performance.

[user@cn3144]$ poretools fastq ERA484348_014370_subset.tar | gzip -c - > 014370.fastq.gz
[user@cn3144]$ ls -lh 014370.fastq.gz
-rw-r--r-- 1 user group 13M Jun 30 08:51 014370.fastq.gz

Create a collector's curve of yield

[user@cn3144]$ poretools yield_plot --plot-type reads --saveas yield.png \
            ERA484348_014370_subset.tar

Read size information

[user@cn3144]$ poretools stats ERA484348_014370_subset.tar
total reads     11787
total base pairs        12470421
mean    1057.98
median  1013
min     320
max     2877
N25     1060
N50     1019
N75     981

[user@cn3144]$ poretools stats --type fwd ERA484348_014370_subset.tar
total reads     3929
total base pairs        4112608
mean    1046.73
median  1001
min     320
max     2833
N25     1044
N50     1007
N75     973

[user@cn3144]$ poretools hist --saveas size.png --theme-bw ERA484348_014370_subset.tar

Quality score distribution by position

[user@cn3144]$ poretools qualpos --saveas qual.pdf \
  --bin-width 100 ERA484348_014370_subset.tar

Exit the interactive session

[user@cn3144]$ exit
salloc.exe: Relinquishing job allocation 46116226
[user@biowulf]$

Create a batch input file (e.g. poretools.sh), which uses the input file 'poretools.in'. For example:

#! /bin/bash
# this file is poretools.batch

module load poretools/0.6.1a1 || exit 1
poretools stats ERA484348_014370_subset.tar > stats
poretools qualpos --saveas qual.png --bin-width 100 ERA484348_014370_subset.tar

Submit this job using the Slurm sbatch command.

sbatch --time=20 --cpus-per-task=2 --mem=6g poretools.sh

Create a swarmfile (e.g. poretools.swarm). For example:

poretools qualpos --saveas qual_s1.png --bin-width 100 s1.tar
poretools qualpos --saveas qual_s2.png --bin-width 100 s2.tar
poretools qualpos --saveas qual_s3.png --bin-width 100 s3.tar

Submit this job using the swarm command.

swarm -f poretools.swarm [-g #] [-t #] --module poretools