Deprecation notice Please note that the functionality in this repository is superseded by integrated pairing in Dorado. This means it's no longer necessary to use pairing or dorado_stereo.sh in order to perform end-to-end duplex calling.

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 Dorado (recommended) and 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.

General Usage

Duplex Tools is run simply with:

duplex_tools --help

The available sub-commands are:

Duplex pairing

Compatible with Dorado

• pair - a wrapper to pair duplex reads, using pairs_from_summary and then filter_pairs.
• split_pairs - a utility for recovering and pairing duplex reads (for cases where template/complement are contained within a single minknow read).

Compatible with Guppy+Dorado

• pairs_from_summary - identify candidate duplex pairs from sequencing summary output by Guppy or unmapped SAM/BAM by dorado.
• filter_pairs - filter candidate pairs using basecall-to-basecall alignment.

Additional tools

• split_on_adapter - split the non-split duplex pairs in to their component simplex reads (formerly read_fillet).
  • This tool splits basecalled sequences into new sequences. For this reason, it's possible to perform basespace duplex calling after using this method, but not regular stereo calling

Usage with Dorado (recommended)

Currently, pairing and calling are separate processes to allow for workflow flexibility.

For greatest duplex recovery, follow these steps:

1) Simplex basecall with dorado (with --emit-moves) 2) Pair reads 3) Duplex-basecall reads

1a) Simplex basecall with dorado

This will create an (unmapped) .sam file which has a mapping between the signal and bases. --emit-moves allows for additional pairs to be found in step 2b.

$ dorado basecaller dna_r10.4.1_e8.2_400bps_fast@v4.0.0 pod5s/ --emit-moves > unmapped_reads_with_moves.bam

2a) Find duplex pairs for Dorado stereo/basespace basecalling

This will detect the majority of pairs and put them in the pairs_from_bam directory.

duplex_tools pair --output_dir pairs_from_bam unmapped_reads_with_moves.bam

2b) Find additional duplex pairs in non-split reads (optional)

The steps below can recover non-split pairs and allows duplex-calling of them.

Use the sam and a pod5 directory to create additional pairs

$ duplex_tools split_pairs unmapped_reads_with_moves.sam pod5s/ pod5s_splitduplex/
$ cat pod5s_splitduplex/*_pair_ids.txt > split_duplex_pair_ids.txt

3) Stereo basecall all the reads

From the main pairing:

$ dorado duplex dna_r10.4.1_e8.2_400bps_sup@v4.0.0 pod5s/ --pairs pairs_from_bam/pair_ids_filtered.txt > duplex_orig.sam

From the additional pairing (optional):

$ dorado duplex dna_r10.4.1_e8.2_400bps_sup@v4.0.0 pod5s_splitduplex/ --pairs split_duplex_pair_ids.txt > duplex_splitduplex.sam

Usage with Guppy

Preparing duplex reads for Guppy duplex 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 dna_r10.4.1_e8.2_400bps_sup.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.