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.

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 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 based pairing run the following:

duplex_tools pairs_from_summary <sequencing_summary.txt> <output directory>

The primary ouput 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>

The first option here is the fle described above and output by pairs_from_summary. The second option should be specified as the Guppy (or MinKNOW) output directory containing fastq data --- the directory will be search recursively for all .fastq.gz and .fastq 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 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 16 \
    --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 files found in the user provided MinKNOW output directory.

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.