dentist 1.0.0

DENTIST

Close assembly gaps using long-reads with focus on correctness.

Today, many genome sequencing project have been conducted using second-generation sequencers which produce short reads. Such assemblies have many gaps. dentist closes these gaps using a (small) set of long reads. Furthermore, it can be used to scaffold contigs freely using a set of long reads. This can be used to fix known scaffolding errors or to further scaffold output of a long-read assembly pipeline.

Table of Contents

• Install
• Usage
• Configuration
• Citation
• Maintainer
• Contributing
• License

Install

Use a Singularity Container (recommended)

Make sure Singularity is installed on your system. You can then use the container like so:

# launch an interactive shell
singularity shell docker://aludi/dentist:latest

# execute a single command inside the container
singularity exec docker://aludi/dentist:latest dentist --version

# run the whole workflow on a cluster using Singularity
snakemake --configfile=snakemake.yml --use-singularity --profile=slurm

The last command is explained in more detail below in the usage section.

Use Pre-Built Binaries

Download the latest pre-built binaries from the releases section and extract the contents. The tarball contains a dentist binary as well as the Snakemake workflow, example config files and this README. In short, everything you to run DENTIST.

Build from Source

Be sure to install the D package manager DUB. Install using either

dub install dentist

or

git clone https://github.com/a-ludi/dentist.git
cd dentist
dub build

Runtime Dependencies

The following software packages are required to run dentist:

• The Dazzler Data Base (>=2020-07-27)

Manage sequences (reads and assemblies) in 4bit encoding alongside auxiliary information such as masks or QV tracks

• DALIGNER (=2020-01-15)

Find significant local alignments.

• DAMAPPER (>=2020-03-10)

Find alignment chains, i.e. sequences of significant local alignments possibly with unaligned gaps.

• DAMASKER (>=2020-01-15)

Discover tandem repeats.

• DASCRUBBER (>=2020-07-26)

Estimate coverage and compute QVs.

• daccord (>=v0.0.17)

Compute reference-based consensus sequence for gap filling.

Please see their own documentation for installation instructions. Note, the available packages on Bioconda are outdated and should not be used at the moment.

Please use the following versions in your dependencies in case you experience troubles. These should be the same versions used in the Dockerfile:

• [email protected]
• [email protected]
• DAZZ_DB@d22ae58
• DALIGNER@c2b47da
• DAMAPPER@b2c9d7f
• DAMASKER@22139ff
• DASCRUBBER@a53dbe8
• [email protected]

Usage

Suppose we have the genome assembly reference.fasta that is to be updated and a set of reads reads.fasta with 25× coverage.

Quick execution with Snakemake (and Singularity)

TL;DR

snakemake --configfile=snakemake.yml --use-singularity --profile=slurm

Install Snakemake version >=5.11.2 and copy these files into your working directory:

• ./snakemake/Snakefile
• ./snakemake/workflow_helper.py
• ./snakemake/snakemake.example.yml → ./snakemake/snakemake.yml

Next edit snakemake.yml to fit your needs and optionally test your configuration with

snakemake --configfile=snakemake.yml -- extend_dentist_config

If no errors occurred the whole workflow can be executed using

snakemake --configfile=snakemake.yml

For small genomes of a few 100 Mbp this should run on a regular workstation. One may use Snakemake's --jobs to run independent jobs in parallel. Larger data sets may require a cluster in which case you can use Snakemake's cloud or cluster facilities.

Executing on a Cluster

To make execution on a cluster easy DENTIST comes with examples files to make Snakemake use SLURM via DRMAA. Please read the documentation of Snakemake if this does not suit your needs. Another good starting point is the Snakemake-Profiles project.

Start by copying these files to your working directory:

• ./snakemake/profile-slurm.yml → ~/.config/snakemake/<profile>/config.yaml
• ./snakemake/cluster.example.yml → ./snakemake/cluster.yml

Next adjust the profile according to your cluster. This should enable Snakemake to submit and track jobs on your cluster. You may use the configuration values specified in cluster.yml to configure job names and resource allocation for each step of the pipeline. Now, submit the workflow to your cluster by

snakemake --configfile=snakemake.yml --profile=<profile>

Note, parameters specified in the profile provide default values and can be overridden by specififying different value on the CLI.

Manual execution

Please inspect the Snakemake workflow to get all the details. It might be useful to execute Snakemake with the -p switch which causes Snakemake to print the shell commands. If you plan to write your own workflow management for DENTIST please feel free to contact the maintainer!

Configuration

DENTIST comprises a complex pipeline of with many options for tweaking. This section points out some important parameters and their effect on the result.

How to Choose DENTIST Parameters

The following list comprises the important/influential parameters for DENTIST itself. Please keep in mind that the alignments generated by daligner/damapper have immense influence on the performance of DENTIST.

• --max-insertion-error: Strong influence on quality and sensitivity. Lower values lead to lower sensitivity but higher quality. The maximum recommended value is 0.05.
• --min-anchor-length: Higher values results in higher accuracy but lower sensitivity. Especially, large gaps cannot be closed if the value is too high. Usually the value should be at least 500 and up to 10_000.
• --reference-error, --reads-error: Determines the -e parameter for daligner/damapper. Use dentist generate-dazzler-options to see the effect of these parameters or consult commandline.d in the source code.
• --min-reads-per-pile-up: Choosing higher values for the minimum number of reads drastically reduces sensitivity but has little effect on the quality. Small values may be chosen to get the maximum sensitivity in de novo assemblies. Make sure to throughly validate the results though.
• --min-spanning-reads: Higher values give more confidence on the correctness of closed gaps but reduce sensitivity. The value must be well below the expected coverage.
• --allow-single-reads: May be used under careful consideration. This is intended for one of the following scenarios:

1. DENTIST is meant to close as many gaps as possible in a de novo assembly. Then the closed gaps must validated by other means afterwards.
2. DENTIST is used not with real reads but with an independent assembly.

• --existing-gap-bonus: If DENTIST finds evidence to join two contigs that are already consecutive in the input assembly (i.e. joined by Ns) then it will preferred over conflicting joins (if present) with this bonus. The default value is rather conservative, i.e. the preferred join almost always wins over other joins in case of a conflict.
• --join-policy: Choose according to your needs:
• scaffoldGaps: Closes only gaps that are marked by Ns in the assembly. This is the default mode of operation. Use this if you do not want to alter the scaffolding of the assembly. See also --existing-gap-bonus.
• scaffolds: Allows whole scaffolds to be joined in addition to the effects of scaffoldGaps. Use this if you have (many) scaffolds that are not yet full chromosome-scale.
• contigs: Allows contigs to be rearranged freely. This is especially useful in de novo assemblies before applying any other scaffolding methods as it increases the contiguity thus increasing the chance that large-scale scaffolding (e.g. Bionano or Hi-C) finds proper joins.

Choosing the Read Type

In the examples PacBio long reads are assumed but DENTIST can be run using any kind of long reads. Currently, this is either PacBio or Oxford Nanopore reads. For using none-PacBio reads, the reads_type in snakemake.yml must be set to anything other than PACBIO_SMRT. The recommendation is to use OXFORD_NANOPORE for Oxford Nanopore. These names are borrowed from the NCBI. Further details on the rationale can found in this issue.

Citation

Arne Ludwig, Martin Pippel, Gene Myers, Michael Hiller. DENTIST – close assembly gaps with high confidence. In preparation.

Maintainer

Dentist is being developed by Arne Ludwig <ludwig@mpi-cbg.de> at the Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.

Contributing

Contributions are warmly welcome. Just create an issue or pull request on GitHub. If you submit a pull request please make sure that:

• the code compiles on Linux using the current release of dmd,
• your code is covered with unit tests (if feasible) and
• dub test runs successfully.

It is recommended to install the Git hooks included in the repository to avoid premature pull requests. You can enable all shipped hooks with this command:

git config --local core.hooksPath .githooks/

If you do not want to enable just a subset use ln -s .githooks/{hook} .git/hooks. If you want to audit code changes before they get executed on your machine you can you cp .githooks/{hook} .git/hooks instead.

License

This project is licensed under MIT License (see LICENSE).