Kieser et al.

BMC Bioinformatics (2020) 21:257

https://doi.org/10.1186/s12859-020-03585-4

SOFTWARE Open Access

ATLAS: a Snakemake workflow for

assembly, annotation, and genomic
binning of metagenome sequence data
Silas Kieser1,2†, Joseph Brown3,4†, Evgeny M. Zdobnov2,5,6, Mirko Trajkovski1,5,7 and Lee Ann McCue3*

* Correspondence: leeann.mccue@
pnnl.gov Abstract
†
Silas Kieser and Joseph Brown
contributed equally to this work. Background: Metagenomics studies provide valuable insight into the composition
3
Earth and Biological Sciences and function of microbial populations from diverse environments; however, the data
Directorate, Pacific Northwest processing pipelines that rely on mapping reads to gene catalogs or genome
National Laboratory, Richland, WA
99352, USA databases for cultured strains yield results that underrepresent the genes and
Full list of author information is functional potential of uncultured microbes. Recent improvements in sequence
available at the end of the article assembly methods have eased the reliance on genome databases, thereby allowing
the recovery of genomes from uncultured microbes. However, configuring these
tools, linking them with advanced binning and annotation tools, and maintaining
provenance of the processing continues to be challenging for researchers.
Results: Here we present ATLAS, a software package for customizable data processing
from raw sequence reads to functional and taxonomic annotations using state-of-the-
art tools to assemble, annotate, quantify, and bin metagenome data. Abundance
estimates at genome resolution are provided for each sample in a dataset. ATLAS is
written in Python and the workflow implemented in Snakemake; it operates in a Linux
environment, and is compatible with Python 3.5+ and Anaconda 3+ versions. The
source code for ATLAS is freely available, distributed under a BSD-3 license.
Conclusions: ATLAS provides a user-friendly, modular and customizable Snakemake
workflow for metagenome data processing; it is easily installable with conda and
maintained as open-source on GitHub at https://github.com/metagenome-atlas/atlas.
Keywords: Metagenomics, Analysis workflow, Annotation, Metagenome-assembled
genomes

Background
Metagenomics has transformed microbial ecology studies with the ability to generate
genome sequence information from environmental samples, yielding valuable insight
into the composition and functional potential of natural microbial populations from di-
verse environments [1, 2]. Despite the prevalence of metagenome data, there are few
broadly accepted standard methods, either for the generation of that data [3–5] or for
its processing [6, 7]. In particular, processing metagenome data in an efficient and
© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to
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permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright
holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless
otherwise stated in a credit line to the data.
Kieser et al. BMC Bioinformatics (2020) 21:257 Page 2 of 8

reproducible manner is challenging because it requires implementation of several dis-

tinct tools, each designed for a specific task.
The most direct and frequently used way to analyze metagenome data is to map the se-
quence reads to reference genomes, when a suitable genome database from cultivated mi-
crobes is available (e.g. Humann2 [8]). However, these methods do not capture
uncultivated species; studies using single-copy phylogenetic marker genes have improved
estimates of species richness in metagenome data by expanding the representation of un-
cultivated species [9]. To truly characterize a natural microbial community and examine
its functional potential, assembly-based metagenome analyses are needed. This has been
demonstrated by recent studies that have recovered thousands of new genomes using co-
abundance patterns among samples to bin contigs into clusters [10–13].
A number of assembly-based metagenome pipelines have been developed, each pro-
viding a subset of the required tools needed to carry out a complete analysis process
from raw data to annotated genomes [14–17]. For example, MOCAT2 [16] relies on
gene catalogs to evaluate the functional potential of the metagenome as a whole, but
without directly relating functions to individual microbes. Metagenome processing
pipelines commonly default to co-assembly of the samples rather than assembly of indi-
vidual samples, resulting in more fragmented assemblies [18]. Only some applications
(e.g., IMP [17]) permit the co-assembly of metagenomes and metatranscriptomes for
individual samples. Furthermore, the configuration and technical constraints to user
control often limit the adoption of these tools in the research community.
Here we present an entirely new version of ATLAS [19], an assembly-based pipeline
for the recovery of genes and genomes from metagenomes, that produces annotated
and quantified genomes from multiple samples in one run with as little as three com-
mands. The pipeline integrates state-of-the art tools for quality control, assembly and
binning. The installation of ATLAS is automated: it depends only on the availability of
Anaconda and installs all dependencies and databases on the fly. The internal use of
Snakemake [20] allows efficient and automated deployment on a computing cluster.

Implementation
The ATLAS framework organizes sequence data processing tools into four distinct ana-
lysis modules: [1] quality control, [2] assembly, [3] genome binning and [4] annotation
(Fig. 1); each module can be run independently, or all four modules combined in a
complete analysis workflow. ATLAS is implemented in Python and uses the Snakemake
[20] workflow manager for extensive control of external tools, including versioning of
configurations and environments, provenance capabilities, and scalability on high-
performance computing clusters. ATLAS uses Anaconda [21] to simplify initial deploy-
ment and environment set-up, and dependencies are handled by Bioconda [22] at run-
time. Complete usage and user options are outlined in the ATLAS documentation
(https://metagenome-atlas.rtfd.io).

Quality control
Quality control of raw sequence data, in the form of single- or paired-end FASTQ files,
is performed using utilities in the BBTools suite [23]. Specifically, clumpify is used re-
move PCR duplicates and compress the raw data files, followed by BBduk to remove
Kieser et al. BMC Bioinformatics (2020) 21:257 Page 3 of 8

Fig. 1 The ATLAS workflow. This high-level overview of the protocol captures the primary goal of the sub-
commands that can be executed by the workflow. Individual modules can be accessed via the command
line or the entire protocol can be run starting from raw sequence data in the form of single- or paired-end
FASTQ files

known adapters, trim and filter reads based on their quality and length (respectively), and
error-correct overlapping paired-end reads where applicable. BBSplit is used to remove
contaminating reads using reference sequences: PhiX is provided as a default or can be re-
placed by user-specified fasta-format sequences. To optimize data use, reads that lose
their mate during these steps are seamlessly integrated into the later steps of the pipeline.

Assembly
Prior to metagenome assembly, ATLAS uses additional BBTools utilities [23] to per-
form an efficient error correction based on k-mer coverage (Tadpole) and paired-end
read merging (bbmerge). If paired-end reads do not overlap, bbmerge can extend them
using read-derived overlapping k-mers. ATLAS uses metaSPAdes [24] or MEGAHIT
Kieser et al. BMC Bioinformatics (2020) 21:257 Page 4 of 8

[25, 26] for de novo assembly, with the ability to control parameters such as k-mer
lengths and k-mer step size for each assembler, as well as hybrid-assembly of paired
short- and long-read libraries. The quality-controlled reads are mapped to the assem-
bled contigs, and bam files are generated to facilitate downstream calculations that may
be of interest (e.g., calculating contig coverage). The assembled contigs shorter than a
minimal length, or without mapped reads, are filtered out to yield high-quality contigs.

Genome binning
The prediction of metagenome-assembled genomes (MAGs) allows organism-specific
analyses of metagenome datasets. In ATLAS, two binning methods are implemented
(Fig. 1): metabat2 [27] and maxbin2 [28]. These methods use tetra-nucleotide frequen-
cies, differential abundance, and/or the presence of marker genes as criteria. ATLAS
supports assembly and binning for each sample individually, which produces more con-
tinuous genomes than co-assembly [29]. Definition of which samples are likely to con-
tain the same bacterial species, via a group attribute in the Snakemake configuration
file, supports binning based on co-abundance patterns across samples. Reads from all
of the samples defined in a group are then aligned to the individual sample assemblies,
to obtain the co-abundance patterns needed for efficient binning. The bins produced
by the different binning tools can be combined using the dereplicate, aggregate and
score tool (DAS Tool, [30]), to yield MAGs for each sample. Finally, the completeness
and contamination of each MAG are assessed using CheckM [31].
Because the same genome may be identified in multiple samples, dRep [29] is used to
obtain a non-redundant set of MAGs for the combined dataset by clustering genomes to
a defined average nucleotide identity (ANI, default 0.95) and returning the representative
with the highest dRep score in each cluster. dRep first filters genomes based on genome
size (default > 5000 bp) and quality (default > 50% completeness, < 10% contamination),
then clusters the genomes using Mash [32], followed by MUMmer [33], thereby benefit-
ting from their combined speed (Mash) and accuracy (MUMmer). The abundance of each
genome can then be quantified across samples by mapping the reads to the non-
redundant MAGs and determining the median coverage across each the genome.

Taxonomic and functional annotation

For annotation, ATLAS supports the prediction of open reading frames (ORFs) using
Prodigal [34]. The translated gene products are then clustered using linclust [35] or
mmseqs [36] to generate non-redundant gene and protein catalogs, which are mapped
to the eggNOG catalogue v5 [37, 38]. Robust taxonomic annotation is performed using
the genome taxonomy database tool kit (GTDB-tk, [39]). In addition, phylogenetic trees
are built based on the markers from GTDB and CheckM.

Output
The ATLAS output for each sample includes the quality-controlled reads, assem-
bled contigs, bam files (reads mapped to contigs), and predicted genome bins, to-
gether with summary statistics in an HTML report. The final output includes
results from all samples, including the raw and normalized counts for the set of
non-redundant, high-quality MAGs, with a quality report and their inferred
Kieser et al. BMC Bioinformatics (2020) 21:257 Page 5 of 8

taxonomy. From the annotation stage, two fasta files are produced containing the
nucleotide and amino acid sequences of the representative genes in the non-
redundant gene catalog, together with a table containing the gene annotations
summarized at the genome level.
Figure 2 shows examples of ATLAS output in which we analyzed the metagenome
data from paired feces and cecum samples of 8 mice fed ad libitum (PRJNA480387
[40];). On average, the sample data contained 3.5 Gbp, and produced assemblies of 108
Mbp per sample. There were 374 MAGS predicted (completeness > 50% and contamin-
ation < 10%), that formed 69 non-redundant clusters (ANI > 99%; Fig. 2A). These ge-
nomes account for 75% of the reads (Fig. 2B). In general, Bacteroides were more
abundant than Firmicutes, in both cecum and feces (Fig. 2C,D). A principal coordinates
analysis based on the functional annotation revealed two functionally distinct clusters
of Firmicutes (Fig. 2E). Details of these results are provided on GitHub (https://github.
com/metagenome-atlas/supp_data_atlas).

Conclusions
ATLAS is easy to install and provides documented and modular workflows for the ana-
lysis of metagenome data. The internal codes utilized by the workflow are highly

Fig. 2 Example output from the ATLAS workflow. Fecal microbiome data (PRJNA480387 [40];) processed by
ATLAS show: A) the completeness and contamination of dereplicated MAGs, with high-quality genomes
highlighted; B) the fraction of reads mapped to genomes; C) a phylogenetic tree of MAGs with average
abundance in feces and cecum on a centered log2 scale; D) a heatmap of abundance on a centered log2
scale in which MAGs were clustered by phylogenetic distance and samples by Euclidian distance; E) a
principle components analysis of the MAGs based on functional annotation
Kieser et al. BMC Bioinformatics (2020) 21:257 Page 6 of 8

configurable using either a configuration file or via the command line. ATLAS provides
a robust bioinformatics framework for high-throughput sequence data, where raw
FASTQ files can be fully processed into annotated tabular files for downstream analysis
and visualization. ATLAS fills a major analysis gap, namely the integration of tools for
quality control, assembly, binning and annotation, in a manner that supports robust
and reproducible analyses. ATLAS provides these analysis tools in a command-line
interface amenable to high-performance computing clusters.
The source code for ATLAS is distributed under a BSD-3 license and is freely avail-
able at https://github.com/metagenome-atlas/atlas, with example data provided for test-
ing. Software documentation is available at https://metagenome-atlas.rtfd.io, which
describes the installation and use of ATLAS including a Docker container (https://hub.
docker.com/r/metagenomeatlas/atlas).
Availability Project name: ATLAS.
Project home page: https://github.com/metagenome-atlas/atlas
Archived version: https://doi.org/10.1101/737528
Operating system(s): Linux.
Programming language: Snakemake/Python.
Other requirements: Miniconda.
License: BSD-3.
Any restrictions to use by non-academics: None.
Acknowledgements
A portion of the ATLAS framework was developed using PNNL Research Computing resources.

Authors’ contributions
JB and SK developed the software and documentation; EZ, MT and LAM supervised the project; and JB, SK and LAM
wrote the manuscript. All authors read and approved the final manuscript.

Funding
This work was supported by the Microbiomes in Transition Initiative at Pacific Northwest National Laboratory (PNNL)
and conducted under the Laboratory Directed Research and Development Program at PNNL, a multi-program national
laboratory operated by Battelle for the U.S. Department of Energy under Contract DE-AC05-76RL01830. This work was
also supported by funding from the European Research Council (ERC) under the European Union’s Horizon 2020 re-
search and innovation programme (ERC-COG-2018), and the Swiss National Science Foundation Professorship to MT.

Availability of data and materials

Not applicable.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests
The authors declare that they have no competing interests.

Author details
1
Department of Cell Physiology and Metabolism, Faculty of Medicine, Centre Medical Universitaire, 1206 Geneva,
Switzerland. 2Swiss Institute of Bioinformatics, Geneva, Switzerland. 3Earth and Biological Sciences Directorate, Pacific
Northwest National Laboratory, Richland, WA 99352, USA. 4Current address: Department of Human Genetics, University
of Utah, 15 S 2030 E, Salt Lake City, UT 84112, USA. 5Institute of Genetics and Genomics in Geneva (iGE3), University of
Geneva, 1206 Geneva, Switzerland. 6Department of Genetic Medicine and Development, University of Geneva, 1206
Geneva, Switzerland. 7Diabetes Center, Faculty of Medicine, Centre Medical Universitaire, 1206 Geneva, Switzerland.

Received: 23 May 2019 Accepted: 8 June 2020

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