Range of tools to support operations on Duplex Sequencing read pairs.
Project description
Duplex Tools
Duplex Tools contains a set of utilities for dealing with Duplex sequencing data. Tools are provided to identify and prepare duplex pairs for basecalling by Guppy and for recovering simplex basecalls from incorrectly concatenated pairs.
Installation
Duplex Tools is written in Python and can be installed directly from PyPI. We recommend installing Duplex Tools into an isolated virtual environment by following:
python -m venv venv --prompt duplex
. venv/bin/activate
pip install duplex_tools
after which the code tools will be available using the duplex_tools
command.
Usage
Duplex Tools is run simply with:
duplex_tools --help
The available sub-commands are:
- split_on_adapter - split incorrectly concantenate duplex pairs in to their component simplex reads (formerly
read_fillet
). - pairs_from_summary - identify candidate duplex pairs from sequencing summary output by Guppy.
- filter_pairs - filter candidate pairs using basecall-to-basecall alignment.
- pair - a wrapper to
pairs_from_summary
and thenfilter_pairs
. Currently only compatible with dorado
Preparing duplex reads for Guppy basecalling
To prepare reads for duplex calling Duplex Tools provides two programs. The first parses the sequencing summary output by the Guppy basecaller (or the metadata in a .bam or .sam from dorado) in order to generate candidate pairs from examining simple read metrics. The second program analyses the basecalls of candidate reads, checking for similarity.
To run the basic sequencing summary(/bam metadata) based pairing run the following:
duplex_tools pairs_from_summary <sequencing_summary.txt/dorado.bam> <output directory>
The primary output of the above will be a text file named pair_ids.txt
in the
user specified output directory. Although this file can be given to Guppy to perform
duplex calling we recommend running the second basecall-to-basecall alignment
filtering provided by the filter_pairs
command:
duplex_tools filter_pairs <pair_ids.txt> <fastq directory/dorado.bam>
The first option here is the file described above and output by pairs_from_summary
.
The second option should be specified as the Guppy (or MinKNOW), or dorado output directory
containing fastq
or bam
data --- the directory will be search recursively for all .fastq.gz
, .fastq
, and .sam/.bam
files.
The output of this second command will be a file named
pair_ids_filtered.txt
placed alongside the pair_ids.txt
file.
Duplex basecalling with Guppy
The file pair_ids_filtered.txt
as prepared above can be used with the
original .fast5
/.pod5
files produced during a sequencing run in order to calculate
high quality duplex basecalls.
For example,
guppy_basecaller_duplex \
-i <MinKNOW directory> \
-r -s duplex_calls \
-x 'cuda:0' -c q20-fixed-2.0-ft-10M.cfg \
--chunks_per_runner 416 \
--duplex_pairing_mode from_pair_list \
--duplex_pairing_file pair_ids_filtered.txt
will produce duplex basecalls using the read pairs stored in the
pair_ids_filtered.txt
file using .fast5
/.pod5
files found in the user
provided MinKNOW output directory.
Duplex basecalling with Dorado
Please use duplex_tools pair unmapped_dorado.bam
.
This will run both the pairing and pairwise alignment-based filtering to get a pair_ids_filtered.txt that can be passed to dorado.
For more details, see https://github.com/nanoporetech/dorado.
Help
Licence and Copyright
© 2021- Oxford Nanopore Technologies Ltd.
Duplex Tools
is distributed under the terms of the Mozilla Public License 2.0.
Research Release
Research releases are provided as technology demonstrators to provide early access to features or stimulate Community development of tools. Support for this software will be minimal and is only provided directly by the developers. Feature requests, improvements, and discussions are welcome and can be implemented by forking and pull requests. However much as we would like to rectify every issue and piece of feedback users may have, the developers may have limited resource for support of this software. Research releases may be unstable and subject to rapid iteration by Oxford Nanopore Technologies.
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