Kutschera et al.

BMC Bioinformatics (2022) 23:228

https://doi.org/10.1186/s12859-022-04757-0 BMC Bioinformatics

SOFTWARE Open Access

GenErode: a bioinformatics pipeline

to investigate genome erosion in endangered
and extinct species
Verena E. Kutschera1* , Marcin Kierczak2 , Tom van der Valk2, Johanna von Seth3,4,5, Nicolas Dussex3,4,5,
Edana Lord3,4,5, Marianne Dehasque3,4,5, David W. G. Stanton3,4, Payam Emami Khoonsari1, Björn Nystedt2,
Love Dalén3,4,5 and David Díez‑del‑Molino3,4,5*   

*Correspondence:
[email protected]; Abstract
[email protected] Background: Many wild species have suffered drastic population size declines over
1
Department of Biochemistry the past centuries, which have led to ‘genomic erosion’ processes characterized by
and Biophysics, National
Bioinformatics Infrastructure
reduced genetic diversity, increased inbreeding, and accumulation of harmful muta‑
Sweden, Science for Life tions. Yet, genomic erosion estimates of modern-day populations often lack concord‑
Laboratory, Stockholm University, ance with dwindling population sizes and conservation status of threatened species.
Solna, Sweden
2
Department of Cell
One way to directly quantify the genomic consequences of population declines is to
and Molecular Biology, National compare genome-wide data from pre-decline museum samples and modern samples.
Bioinformatics Infrastructure However, doing so requires computational data processing and analysis tools specifi‑
Sweden, Science for Life
Laboratory, Uppsala University,
cally adapted to comparative analyses of degraded, ancient or historical, DNA data with
Uppsala, Sweden modern DNA data as well as personnel trained to perform such analyses.
3
Centre for Palaeogenetics,
Svante Arrhenius väg 20C, 106
Results: Here, we present a highly flexible, scalable, and modular pipeline to compare
91 Stockholm, Sweden patterns of genomic erosion using samples from disparate time periods. The GenErode
4
Department of Bioinformatics pipeline uses state-of-the-art bioinformatics tools to simultaneously process whole-
and Genetics, Swedish Museum
of Natural History, Stockholm,
genome re-sequencing data from ancient/historical and modern samples, and to
Sweden produce comparable estimates of several genomic erosion indices. No programming
5
Department of Zoology, knowledge is required to run the pipeline and all bioinformatic steps are well-docu‑
Stockholm University, 106
91 Stockholm, Sweden
mented, making the pipeline accessible to users with different backgrounds. GenErode
is written in Snakemake and Python3 and uses Conda and Singularity containers to
achieve reproducibility on high-performance compute clusters. The source code is
freely available on GitHub (https://​github.​com/​NBISw​eden/​GenEr​ode).
Conclusions: GenErode is a user-friendly and reproducible pipeline that enables the
standardization of genomic erosion indices from temporally sampled whole genome
re-sequencing data.
Keywords: Bioinformatics, Temporal genomic erosion, Conservation genomics, Whole
genome re-sequencing data, Endangered species, Reproducibility, Snakemake, Ancient
DNA

© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits
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Kutschera et al. BMC Bioinformatics (2022) 23:228 Page 2 of 17

Background
A plethora of large scale projects are set to generate de novo assemblies for all eukaryotic
species in the next few years (e.g. [1–3]), thus solving one of the traditional limitations of
conservation genomic studies, namely the absence of reference genomes for non-model
species. In all these projects, contributing to biodiversity conservation appears among
the main objectives. They propose to do this by enabling genomic studies in endangered
species, which has been sometimes presented as the solution to all sorts of conserva-
tion problems [4, 5]. Yet there are currently only a limited number of examples where
genomic data has had concrete conservation impacts (e.g., [6–8]). While the reasons for
this are several-fold, two of the most relevant ones are existing biases among genomic
studies due to the lack of standard measures, and absence of validation of results using
empirical data [9].
Human activities have caused populations of many wild species to suffer severe
declines over the past few centuries [10]. These dwindling population sizes have led to
an increased risk of extinction [11] as a consequence of genetic processes, such as the
loss of genomic diversity, increased inbreeding, and accumulation of harmful muta-
tions, processes together referred to as ’genomic erosion’ [12–14]. Therefore, genomic
approaches aimed at identifying the role of genomic erosion in shaping the fate of endan-
gered populations are key to implementing more effective conservation efforts. Unfortu-
nately, correlations between genomic erosion indices and current population sizes and/
or the conservation status of endangered species are weak [15], which further compli-
cates the inclusion of genetic parameters as a criterion to establish threat categories.
This is because ancient bottlenecks or life-history traits typically overshadow the impact
of the recent human-driven declines [16]. Consequently, interspecific comparisons of
genome-wide diversity, inbreeding, and mutational load are poor predictors of popula-
tion size and conservation status between modern-day endangered species. One power-
ful solution to this issue is to obtain genomic data from historical or ancient specimens
that predate the recent demographic declines. Such temporal data can then be used to
establish pre-decline baselines and enable direct quantification of the rate of change in
genomic erosion indices that have resulted from recent population declines [15].
A second important limitation to bridging the gap between genomic studies of endan-
gered species and applications in conservation is the need for advanced bioinformatics
knowledge [9]. Handling and analyzing genomic data requires expertise in processing
large-scale DNA datasets and programming skills that, sometimes, conservation biolo-
gists and wildlife managers are lacking. On top of this, the bioinformatic processing of
the data and the interpretation of the analyses can be further complicated by the special
characteristics of degraded historical and ancient DNA. Finally, reproducibility (obtain-
ing the same results from analyzing the same data) is lacking across scientific disciplines
[17]. However, genomic erosion indices can only be meaningful, comparable measures, if
the results are reproducible. Therefore the increased access to user-friendly software and
structured pipelines that generate reproducible results can be key factors for genomic
data to be regularly applied to conservation [18].
With the aim to address these issues, we present GenErode, a highly flexible and
modular pipeline focused on the user-friendly application of genomic data for conserva-
tion genomics. It performs analyses of whole-genome re-sequencing data, enabling the
Kutschera et al. BMC Bioinformatics (2022) 23:228 Page 3 of 17

analysis of temporally sampled genomes with the goal to investigate patterns of genome
erosion. GenErode employs state-of-the-art techniques to bioinformatically process
genomic data and can handle both ancient/historical and modern samples simultane-
ously. It also generates estimates of commonly used genomic erosion indices (e.g.,
genome-wide diversity, inbreeding, and mutational load) with the objective of making
them directly comparable between time periods.

Implementation
Description
In this pipeline, ancient/historical and modern whole-genome re-sequencing data are
mapped to a reference genome assembly and processed according to the characteris-
tics of each data type, aiming to make them comparable among different time periods
(Fig. 1). While it is optimized for the analysis of vertebrate re-sequencing data, all of the
data processing tracks can be applied to a variety of organisms, including, for example,

Fig. 1 Overview of the GenErode pipeline data processing tracks. Input and output file formats,
dependencies between steps, and main software used are shown. Optional steps are highlighted in red
Kutschera et al. BMC Bioinformatics (2022) 23:228 Page 4 of 17

haploid insects. Written in Snakemake [19], this pipeline can be run on Linux systems
such as high-performance computing (HPC) clusters.
In short, GenErode takes paired-end FASTQ files as input, maps them to a refer-
ence genome assembly, performs variant calling, and runs several downstream analyses
to estimate various genomic erosion indices. All data processing steps include quality
checks and filtering. Optional steps include post-mortem damage base quality rescal-
ing of historical/ancient samples, removal of cytosine-guanine dinucleotide (CpG) sites,
subsampling a proportion of reads to achieve a similar average depth across samples,
and mapping of historical/ancient data to human and other vertebrate mitochondrial
genomes to assess the presence of non-endogenous reads in the data.

Data processing track

Each data processing step is dependent on previous pipeline steps and is thus automati-
cally run in sequence (see Fig. 1).

1. Reference genome assembly preparations

Prior to raw data processing, the reference genome assembly is prepared to generate
files required for downstream analyses. This includes indexing with BWA [20] and sam-
tools [21], generation of a FASTA dictionary using Picard (https://​github.​com/​broad​
insti​tute/​picard) and of genome-coordinate BED files from the reference. At this stage,
the pipeline also performs de novo identification and masking of repetitive regions of
the genome using RepeatModeler [22] and RepeatMasker [23]. The identified repetitive
regions will be excluded from all downstream steps of the data analysis track in order to
avoid biases caused by mismapping.

2. Data processing and mapping

Raw FASTQ files are trimmed with fastp [24], which automatically detects adapter
sequences from the read data, under the assumption that only one adapter is present
in the reads. Fastp also automatically enables poly-G trimming for Illumina NovaSeq
or NextSeq samples by checking the flow cell identifier, so it handles read trimming
for multiple Illumina platforms. For historical samples, fastp simultaneously adapter-
and quality-trims reads and merges overlapping paired-end reads. By default, merged
reads below a threshold of 30 bp are discarded. However, this threshold can be modi-
fied using the configuration file. Most ancient and historical DNA studies discard
reads below a threshold of length between 30 and 35 bp, depending on the preserva-
tion of the samples (e.g. [25, 26]). The default merging settings are recommended in
order to exclude modern-day contaminating sequences for which paired-end reads
will not overlap since they are typically longer. Merged reads are then mapped to
the reference genome using BWA aln with settings optimized for ancient/histori-
cal DNA (-l 16500 -n 0.01 -o 2) [27]. For modern samples, FASTQ files are adapter-
and quality-trimmed with fastp and then mapped to the reference genome assembly
with BWA mem [28] using default settings. For ancient/historical samples, multiple
sequencing libraries are commonly generated per sample to avoid overrepresentation
Kutschera et al. BMC Bioinformatics (2022) 23:228 Page 5 of 17

of specific PCR duplicates in the read data, using different index(es) for each sequenc-
ing library. The alignments of each index of a sample that were sequenced on different
lanes are therefore merged to generate one BAM file per index. Next, PCR duplicates
are identified and excluded from ancient/historical data using both read start and end
mapping coordinates using a custom Python script. For modern samples, the same
merging algorithm is run if applicable and PCR duplicates are identified and marked
using Picard MarkDuplicates (). For both historical and modehttps://​broad​insti​tute.​
github.​io/​picard/rn data, the alignments of each sample from different indices are
then merged to generate one BAM file per sample, and reads around indels are rea-
ligned with GATK IndelRealigner [29] to improve mapping accuracy. Basic mapping
statistics are reported for each processing step using samtools and Qualimap [30],
and are summarized using MultiQC [31]. Histograms depicting the depth per site as
well as the genome-wide average depth and minimum and maximum depth thresh-
olds for downstream analyses are also generated. The user can decide if genome-wide
average depth should be calculated including or excluding sites with zero coverage.
By default, depth thresholds are set to 1/3 and ten times the average genome-wide
depth of each sample, which should be adjusted by the users according to their data
characteristics. However, the pipeline uses an absolute minimum depth of three reads
per site that cannot be changed by the user. Although the pipeline can be run with
samples sequenced to lower depths, an average genome-wide depth of at least 6X per
sample is recommended to have enough statistical power for detecting heterozygous
sites. MultiQC reports and depth histograms based on BAM files after indel realign-
ment are included into an automatically generated GenErode pipeline report.
There are two main optional steps to further process the BAM files before running
any downstream data analyses: (1) base quality rescaling with MapDamage2 [32] for
selected ancient/historical samples that have not been treated with the uracil-DNA
glycosylase (UDG) enzyme and show post-mortem damage; and (2) subsampling of
the selected BAM files from ancient/historical and/or modern samples using sam-
tools to a target genome-wide depth to avoid biases introduced when comparing sam-
ples at different coverages. Additionally, a mitochondrial contamination check can be
run for selected ancient/historical samples, in which the trimmed and merged reads
are aligned to the mitogenomes of a set of species for which contamination may be
present in the laboratory (i.e. from the laboratory reagents [33]). The user is asked
to include a mitogenome FASTA file of the target species at this step. The GenErode
pipeline produces BAM file statistics and a table listing the ratio of mapped reads to
the mitochondrial genome of a potentially contaminating species and of the target
species to help identify sequencing libraries with more reads mapping to the mitog-
enome from a different species. The results from this mitochondrial contamination
check are not further used in the pipeline but the BAM files containing the sequences
mapping to the mitochondrial genome of the target species are kept so that they can
be used for downstream analyses outside of the pipeline. After each of these optional
steps, basic mapping statistics are reported using samtools and Qualimap, and are
summarized using MultiQC.
Kutschera et al. BMC Bioinformatics (2022) 23:228 Page 6 of 17

3. Genotyping and variant filtering

This part of the pipeline is designed to perform variant calling on a per-sample basis. The
rationale being that ancient, historical, and modern samples typically come from disparate
time periods and locations, so using the information from some samples to predict vari-
ation in others can lead to undesired biases. Therefore, variants are called in each sample
using bcftools [34] mpileup and call. Before proceeding to downstream analyses, the vari-
ant calls are subjected to several filtering steps. Methylated CpG sites are protected from
UDG enzyme treatments and so post-mortem damage might remain in such sites [35, 36].
Ideally, a variant filter should therefore capture sites in ancient/historical samples that have
a basepair change from CpG due to post-mortem damage. GenErode offers three optional
methods to identify CpG sites: (1) identifying them in the reference genome assembly, (2)
identifying them in selected samples once genotyped; and (3) combining both strategies.
In all three cases, these CpG sites are automatically excluded from the final VCF files and
downstream analyses. For datasets with only one or a few samples, we recommend identify-
ing CpG sites in the reference genome assembly (option 1), which will remove damaged (or
mutated) sites in the samples that are CpG in the reference. In larger datasets composed of
modern and ancient/historical samples that are mapped to a more distantly related refer-
ence genome assembly (e.g. from a different species than the samples), it is recommended
to identify CpG sites using all genotyped samples (option 2). Finally, we recommend a com-
bination of both strategies (option 3) when a more stringent CpG filter is desired. After CpG
site removal from VCF files, the pipeline moves on to other filtering steps. Only sites with
mapping and base qualities of at least 30 are kept. The pipeline will exclude all variants that
are located within 5 bp of indels (insertions or deletions), and will subsequently remove all
indels. Also, all sites falling outside the depth thresholds specified by the user (as described
above) are removed. An allelic imbalance filter removes heterozygous sites in which less
than 20% or more than 80% of reads support each allele to avoid erroneous genotypes
caused by contamination or misalignments [37]. Variants falling within repetitive regions,
earlier identified in the reference genome assembly, are also excluded in this step.
Finally, VCF files from all samples are merged and sites that are not biallelic as well as
sites with more than 10% missing genotypes across all samples are removed. This miss-
ingness threshold can be adjusted by the user to suit each particular dataset, for example
by relaxing it when many samples are to be analyzed at the same time. A BED file is cre-
ated of all the genomic locations of sites remaining after the filtering and the merged
VCF file is split into one VCF file of historical and one VCF file of modern samples.

Data analysis track

All steps in the data analysis tracks are optional and can be run independently from each
other once the data processing track is finished (see Fig. 2).

4. Genome-wide diversity

GenErode uses mlRho [38] to estimate the maximum likelihood population muta-
tion parameter (θ = 4Neμ), which under the infinite sites model approximates the per-
site heterozygosity [39], from the final BAM file of each sample from step 2. All sites
Kutschera et al. BMC Bioinformatics (2022) 23:228 Page 7 of 17

Fig. 2 Overview of the GenErode pipeline data analysis tracks and final report. Input file formats and main
software used are shown

are filtered for mapping and base qualities as described above and for depth of cover-
age using the specified minimum and maximum thresholds. All sites within repetitive
regions are excluded by default and CpG sites can be excluded if desired. GenErode also
allows to estimate heterozygosity for autosomes and sex chromosomes separately if a
list of known sex chromosomes (or scaffolds) is provided. BED files of repeat elements
and/or CpG sites on autosomes and sex chromosomes are automatically generated using
BEDtools [40]. A table and a figure summarizing θ estimates from mlRho are provided
in the GenErode pipeline report.

5. Inbreeding

Based on the VCF file of filtered genotypes from step 3, the GenErode pipe-
line uses the sliding window approach of ‘plink –homozyg’ [41] to identify runs of
Kutschera et al. BMC Bioinformatics (2022) 23:228 Page 8 of 17

homozygosity (ROH). A wide array of plink settings such as ROH minimum size,
maximum number of heterozygous sites per window, and maximum number of miss-
ing sites per window can be specified by the user. Long ROH (> = 2 Mb) typically arise
from recent mating with close relatives [42]. The fraction of the genome of each sam-
ple allocated in such ROH can be defined as the inbreeding coefficient (FROH), which
is estimated and visualized in a plot that is provided in the GenErode pipeline report.

6. Population structure

The general population structure for the samples analyzed in the pipeline can be visu-
alized with a Principal Component Analysis (PCA). Based on the VCF file of filtered
genotypes from step 3, the pipeline generates PCAs for all samples combined and for
historical and modern samples alone using plink. This analysis can be used to check
genetic population structure, as well as to identify genetic outliers or biases among
samples. All the PCA plots are made available in the GenErode pipeline report.

7. Mutational load

GenErode offers two complementary methods to estimate mutational load, a proxy

for genetic load [43], from the genomic data of the samples analyzed. For users
intending to apply these two methods in endangered species for which the only
remaining population is small or very small, we recommend running the GenErode
pipeline using the reference genome assembly of a closely related species rather than
the same species as the samples [43]. This is to avoid biases caused by a functional
annotation that is based on an already genetically depauperate sample or from calling
variants from data mapped to a reference genome assembly individual that is more
genetically related to some of the samples under comparison (i.e., more related to the
modern samples than to the historical ones).
The first method requires a genome annotation in GTF2 format based on which
GenErode annotates individual variants from step 3 using snpEff [44]. snpEff allows to
annotate all identified variants per sample and to estimate a simple prediction of their
functional effects which can be compared among samples or groups of samples (i.e.,
historical vs modern). MultiQC reports summarizing snpEff results per dataset are
provided in the GenErode pipeline report. Numbers of variants of different impact
categories per sample (including loss-of-function (LOF) variants in the high impact
category) as estimated by snpEff are visualized in a plot that is also available in the
GenErode pipeline report.
The second method uses GERP scores [45] to infer the relative mutational load of
each sample from the number of derived alleles in evolutionary constrained regions
of the genome based on a panel of outgroup species [46]. This method does not
require a functional annotation for the target species’ reference genome assembly, but
it does require at least 30 genome assemblies from different species to act as out-
groups [46], and a dated phylogeny from all the included species in NEWICK format
and in billions of years (which can be obtained from, e.g. timetree.org). Our pipeline
first splits each outgroup genome assembly into non-overlapping 35 bp sequences
Kutschera et al. BMC Bioinformatics (2022) 23:228 Page 9 of 17

and maps them to the target species reference genome assembly to identify highly
conserved regions of the genome [46]. Next, it estimates a per-site GERP score using
GERP++ [47]. Finally, the ancestral and derived states of each variant from step 3
are inferred and used to estimate a relative mutational load. For each sample, het-
erozygous and homozygous sites are counted as contributing to one and two derived
alleles, respectively. Only derived sites with a GERP score within a minimum and a
maximum threshold set by the user are considered for relative load mutational cal-
culations. Our approach is not suitable to identify fast evolving regions and positive
GERP scores indicate evolutionary constraint, so a suitable minimum GERP score of
at least zero should be chosen. A script to approximate a minimum GERP score based
on a desired percentile of GERP scores from the genome from histogram bins is avail-
able in the GenErode GitHub repository (‘https://​w ww.​utili​ties/​get_​gerp_​score_​perce​
ntile.​py’). A histogram of GERP scores across the genome is available in the GenErode
pipeline report and can be used as guidance. At each of these sites, the GERP score
is multiplied by the number of derived alleles per sample. The sum of these GERP
scores is then normalized by the number of derived alleles at these sites (modified
from [46]), to obtain a relative mutational load estimation for each sample. Finally,
the pipeline generates a table and a figure with the relative mutational load estimates
across samples that are included in the GenErode pipeline report.

Test dataset
To help the user get familiarized with our pipeline we provide a test dataset based on
the Sumatran rhinoceros (Dicerorhinus sumatrensis) re-sequencing data from von Seth
et al. [43]. The test dataset is composed of re-sequencing data from three historical
and three modern samples from the now-extinct Malay Peninsula population (Addi-
tional file 1: Table S1), which had been analyzed for patterns of genome erosion. These
data correspond to those sequences that mapped to a single scaffold of 41 Mb size
(‘Sc9M7eS_2_HRSCAF_41’) from the Sumatran rhinoceros genome assembly (Gen-
Bank accession number GCA_014189135.1), as well as to the mitochondrial genome
(GenBank accession number NC_012684.1), which are both provided as references. A
small proportion of sequences mapping elsewhere in the genome were included too. We
have also included three scaffolds from the white rhinoceros genome assembly (Cera-
totherium simum simum; GenBank accession number GCF_000283155.1) for analy-
ses that require a reference from a more distantly related species (‘NW_004454182.1’,
‘NW_004454248.1’, and ‘NW_004454260.1’). These scaffolds had been identified as being
putatively orthologous to the Sumatran rhinoceros scaffold ‘Sc9M7eS_2_HRSCAF_41’.
For mutational load analyses, gene predictions from the three white rhinoceros scaffolds
were extracted in GTF format and scaffolds from the genome assemblies of 30 mamma-
lian outgroup species (Additional file 1: Table S2) were identified as putative orthologs
to the Sumatran rhinoceros scaffold. A phylogeny of the white rhinoceros and the 30
outgroup species including divergence time estimates (in billions of years) was obtained
from timetree.org in NEWICK format (Additional file 1: Fig. S1). A detailed description
of the test dataset generation is provided in Additional file 1: Extended methods note 1.
The test dataset is publicly available at the Scilifelab Data Repository (https://​doi.​org/​10.​
17044/​scili​felab.​19248​172). The results presented in Fig. 3 have been generated using the
Kutschera et al. BMC Bioinformatics (2022) 23:228 Page 10 of 17

Fig. 3 Results from the downstream analyses for historical and modern Sumatran rhinoceros samples
from the test dataset. a Principal component analysis (PCA) of historical and modern Sumatran rhinoceros
samples using the Sumatran rhinoceros reference. Historical samples: JvS008, JvS009, JvS022. Modern
samples: JvS033, JvS034, JvS035. See “PCA” in Additional file 3. b Maximum likelihood estimates of
genome-wide heterozygosity (θ) and 95% confidence intervals from mlRho in historical and modern
Sumatran rhinoceros samples using the Sumatran rhinoceros reference. See “mlRho” in Additional file 3.
c Inbreeding coefficient estimated as the proportion of the genome in runs of homozygosity (FROH) for
ROH of length >  = 2 Mb in historical and modern Sumatran rhinoceros samples. Parameter settings in the
configuration file: homozyg-snp: 25; homozyg-kb: 100; homozyg-window-snp: 250; homozyg-window-het: 3;
homozyg-window-missing: 15; homozyg-het: 750. See “ROH” in Additional file 3. d Number of variants in each
impact category in historical and modern Sumatran rhinoceros samples as estimated by snpEff and using
the white rhinoceros reference with 253 protein-coding gene annotations. Note that none of the samples
contained any high impact variants which are therefore not shown. See “snpEff” in Additional file 2. e Relative
mutational load estimated from GERP scores for historical and modern Sumatran rhinoceros samples using
the white rhinoceros reference. GERP scores were estimated using 30 outgroup species from across the
tree of mammals. Only derived alleles from sites with GERP scores above the 99th percentile were included
(corresponding to GERP > 0.00048928). See “GERP” in Additional file 2

GenErode pipeline for this dataset on a HPC cluster using a slurm workload manager.
The commands used to set up and run the pipeline to analyze the test dataset are pro-
vided in Additional file 1: Extended methods note 2. The final reports generated by the
Kutschera et al. BMC Bioinformatics (2022) 23:228 Page 11 of 17

GenErode pipeline are provided as Additional file 2 and Additional file 3, which include
configuration files specifying all parameter choices.

Results and discussion

Requirements and pipeline configuration
For a species with a genome size of around 3 Gbp the GenErode pipeline requires a
Linux system with at least 16 cores, each with a minimum of 6 GB of RAM (a total of
96 GB), and internet access so Snakemake can download the required containers on the
fly. Depending on the number of samples to analyze and the sequencing effort of each
sample the storage space required could be large. For example, in a test run with four
Sumatran rhinoceros samples with an average genome-wide depth ranging from 9 to
18X and mapped to the Sumatran rhinoceros genome assembly, the pipeline directory
size after running all data processing and downstream analyses (except the storage-space
demanding GERP step) had a final size of ca. 750 GB. However, this was after GenEr-
ode had removed temporary intermediate files and the space required reached several
TB at some points during this pipeline run, so storage space flexibility is recommended.
The only software requirements to run the pipeline are Singularity (at least v3.6.1) and
Conda (tested with Miniconda3), which have to be installed on the system. The GenEr-
ode pipeline is installed by cloning the GitHub repository into the directory in which
it will be run. The user needs to create a Conda environment from the ‘environment.
yml’ file provided in the GitHub repository, which contains Snakemake (version 6.12.1
[19]), Python3, and all required additional python modules. All custom python scripts
used by the pipeline are included in the GitHub repository and all additional software
and dependencies are automatically downloaded as Docker images and run as Singular-
ity containers by Snakemake, so there is no need for any additional installations by the
user. All output files of the pipeline will be automatically written to subdirectories within
the GenErode directory, except for any files related to the reference genome assembly
and the genome annotation, which will be written in the same directory as the reference
genome assembly.
To configure a pipeline run, the user first needs to create a metadata file in which the
sample names, sequencing runs, read group IDs, and paths to the raw sequencing files
are specified. Examples are available in the ‘config’ directory of the GitHub repository
and in the Scilifelab Data Repository containing the test dataset (see below). This pipe-
line has been developed using the slurm workload manager [48] with the official Snake-
make profile for slurm (https://​github.​com/​Snake​make-​Profi​les/). The computational
resources for the different jobs that are submitted to the cluster via Snakemake are spec-
ified in a cluster configuration file (‘config/cluster.yaml’) that needs to be edited accord-
ing to the user’s system. Finally, to configure each run of the pipeline the user needs to
edit the pipeline configuration file (‘config/config.yaml’) in which each step of the pipe-
line can be set to ‘True’ or ‘False’. Additionally, the user can specify any required param-
eters for the desired steps to run. For example, the adapter trimming step for historical
samples requires the user to specify the minimum read length allowed after trimming
and read merging (default 30 bp). When a step is set to ‘True’, all analyses within this
step (‘rules’) are automatically run along with any upstream rules they depend on. For
Kutschera et al. BMC Bioinformatics (2022) 23:228 Page 12 of 17

example, if the mapping step is set to ‘True’, adapter trimming and quality controls of
raw and trimmed reads are also automatically run in case these previous rules have not
been run before.
A complete guide on the requirements and configuration of the pipeline, including
detailed explanations for each step and the parameters, is available in the wiki section of
the pipeline’s GitHub page (https://​github.​com/​NBISw​eden/​GenEr​ode/​wiki/).

Test dataset: genomic erosion in the Sumatran rhinoceros

We analyzed the test dataset of re-sequencing data from three historical and three mod-
ern Sumatran rhinoceros samples from the now-extinct Malay Peninsula population
(Additional file 1: Table S1; see “Implementation: Test dataset” and Additional file 1:
Extended methods note 1). The GenErode pipeline was run once using the Sumatran
rhinoceros scaffold ‘Sc9M7eS_2_HRSCAF_41’ as reference to obtain PCAs, θ and FROH
estimates, and a second time using the putatively orthologous white rhinoceros scaffolds
‘NW_004454182.1’, ‘NW_004454248.1’, and ‘NW_004454260.1’ as reference to infer
PCAs and mutational load using snpEff annotations and GERP scores. The Sumatran
rhinoceros scaffold consists of 40.8 Mbp of which 27.7% were masked by RepeatMas-
ker. The white rhinoceros scaffolds have a combined length of 41.2 Mbp of which 27.1%
were repeat masked. Three to 24 sequencing libraries were available per historical sam-
ple; each sample had been sequenced on two Illumina HiSeqX lanes. For each modern
sample, one sequencing library was included. An average number of ca. 4.6 M paired-
end reads were included into the test dataset per historical sample, and ca. 3.1 M paired-
end reads were included per modern sample. The optional GenErode step to test for
overrepresentation of mitochondrial reads in historical libraries revealed ratios of reads
mapping to the included mitochondrial genomes (see Additional file 1: Table S3) and
the Sumatran rhinoceros mitochondrial genome ranging from 0.0 to 0.14 (mean = 0.052,
sd = 0.037). This suggests that the presence of non-endogenous reads in the historical
libraries of Sumatran rhinoceros is low or absent.
For the historical samples, after filtering per index and sample and PCR duplicate
removal, an average of 2.7 M and 2.2 M reads mapped to the Sumatran and white rhi-
noceros references, respectively. Average genome-wide depth ranged from 4 to 6 for the
Sumatran rhinoceros reference and 4 to 5 for the white rhinoceros reference (Additional
file 1: Fig. S2A). For the modern samples, after filtering and PCR duplicate marking, an
average of 3.0 M and 2.9 M paired-end reads mapped to the Sumatran and white rhinoc-
eros references, respectively. This corresponds to average genome-wide depths ranging
from 16x to 20x across all samples and references (Additional file 1: Fig. S2B). Prior to
downstream data analysis steps, BAM files from modern samples were subsampled to an
average depth of 6. In total, 397,642 and 395,780 CpG sites were identified in the Suma-
tran and white rhinoceros references, respectively. These sites were removed from indi-
vidual VCF files followed by subsequent filtering using default settings of the pipeline.
The filtered and merged multi-sample VCF files contained 7,484 SNPs for the Sumatran
rhinoceros reference and 40,352 SNPs for the white rhinoceros reference. This difference
can be explained with a large number of sites where the Sumatran rhinoceros samples
Kutschera et al. BMC Bioinformatics (2022) 23:228 Page 13 of 17

are homozygous for the ALT allele, i.e. they are diverged from the white rhinoceros
reference.
In a PCA of Sumatran rhinoceros samples using the VCF file based on the Sumatran
rhinoceros reference, the first principal component (PC1) explained 31.8% of the vari-
ation (Fig. 3a). The three modern samples clustered slightly closer together than the
three historical samples. A PCA using the VCF file based on the white rhinoceros ref-
erence resulted in a highly similar plot (Additional file 1: Fig. S3). GenErode was next
run to estimate various genomic erosion indices to compare them between historical
and modern samples. Genome-wide heterozygosity was estimated as θ in mlRho using
BAM files based on the Sumatran rhinoceros reference with default filtering settings
and excluding CpG sites. Although heterozygosity for the test scaffold was lower than
for the whole genome [43], historical and modern samples were similar (Fig. 3b), con-
sistent with genome-wide temporal comparisons from [41]. ROH were estimated using
the VCF file based on the Sumatran rhinoceros reference (see “ROH” and “Configura-
tion” in Additional file 3 for parameter choices). Larger proportions of the reference
were in ROH >  = 2 Mb in modern samples than in historical samples (Fig. 3c), suggest-
ing stronger effects of inbreeding in the modern population, consistent with von Seth
et al. [43]. No variants of potentially high impact on the protein (which include LOF
variants) were found in any of the samples in the snpEff analyses of the VCF files based
on the white rhinoceros reference and 253 gene predictions. Numbers of variants in the
other impact categories were similar across historical and modern samples (Fig. 3d).
Unlike genome-wide estimates [43], relative mutational load estimated from the top 1%
of GERP scores based on the white rhinoceros reference and 30 outgroup species (Addi-
tional file 1: Table S2, Fig. S1) were similar between historical and modern samples for
our test dataset (Fig. 3e; see Additional file 1: Fig. S4 for a histogram of GERP scores).
The expected runtime of all jobs run by GenErode for this test dataset on a HPC clus-
ter with 16 cores (6 GB of RAM per core) can be up to ca. 15 core hours for data pro-
cessing and downstream analyses using the Sumatran rhinoceros reference, and ca. 47
core hours for data processing and downstream analyses using the white rhinoceros
reference. The results from the original genome-wide dataset, generated with an under
development version of the GenErode pipeline, can be consulted in [43].

A bioinformatics pipeline for ancient/historical, and modern data

There are established bioinformatic pipelines tailored to processing and genotyping
ancient DNA data (e.g. [49]) as well as mining metagenomic information from it (e.g.
[50]). However, to our knowledge, GenErode is the first pipeline specifically designed
to process ancient, historical and modern sequencing data from the same species with
the aim to make them comparable. This is because GenErode has separately optimized
tracks for processing of ancient/historical and modern data, enabling a better com-
parison between samples from different time periods. Moreover, in its simplest con-
figuration, GenErode requires only a reference genome assembly and whole-genome
re-sequencing data. It is therefore highly suitable for analyses of data from non-model
species that often lack genomic resources, which is often the case for endangered taxa.
Our pipeline also outputs the results of a number of downstream analyses including
estimates of genomic diversity indices, such as heterozygosity and inbreeding, as well
Kutschera et al. BMC Bioinformatics (2022) 23:228 Page 14 of 17

as mutational load (including per-sample LOF estimates), that can be used to quantify
genomic erosion through time. These genomic erosion indices have been proposed
to be relevant in assessing the species risk of extinction when compared among time
periods, such as historical versus modern samples (e.g., [15, 43, 51, 52]). It should be
noted that GenErode offers only one estimation method for each data analysis track
and genomic erosion index. While this can be sufficient to draw conclusions for some
studies, for users that desire to conduct a more in-depth analysis of their data, the
output files produced (i.e. BAM and VCF files) can be easily used as input for other
types of analyses outside of the pipeline.
One important feature of GenErode is that it is a highly modular and flexible pipe-
line. It allows analyzing the data in a stepwise fashion with important options pre-set
for the user but also plenty of customizable parameters and several optional steps so
the user can adapt it to their particular dataset. Even though the genotyping and data
analysis tracks are specifically designed for samples sequenced at a minimum average
genome-wide depth of 6X, the data processing tracks (read processing, mapping, and
BAM file filtering) can be used for samples of all depths. This means that the pipeline
can also process low or very low coverage samples, either UDG treated or not, typi-
cal for ancient DNA projects or museum collections with highly degraded specimens,
and at the same time provide tools for authentication such as post-mortem damage
profiles.
Finally, alongside a comprehensive variety of output result files, including an auto-
matically generated pipeline summary report, GenErode stores an organized set of
parameter files and output logs for each step, providing the user with all information
required to thoroughly document their analysis. Also, the majority of the pipelines’
analyses are run in separate, automatically executed Singularity containers, which
enables the user to process and analyze sequencing data in a highly reproducible way
[53]. Reproducibility still remains a challenge for most bioinformatic applications and
analyses from published articles across the sciences [17], but is key to obtaining com-
parable genomic erosion measurements. This is necessary to integrate them into the
criteria to establish threat categories in endangered species’ lists [8].

Conclusions
We introduce GenErode, a bioinformatic pipeline to investigate genomic erosion that
includes state-of-the-art tools for ancient/historical, and modern DNA data within an
integrated, easily reproducible and accessible format. Two of the main challenges to
close the gap between evolutionary research and conservation applications are the lack
of standard methods for the measurement of genomic diversity, as well as the need for
training in the bioinformatic methods required to analyze genomic data. GenErode aims
to produce comparable estimates of genomic diversity indices from temporally sampled
datasets that can be used to quantify genomic erosion through time. Additionally, set-
ting up and running GenErode requires no programming knowledge and all the bio-
informatic steps are well documented, making it easy to run for people with different
backgrounds. Therefore, with this pipeline we aim to make the bioinformatic methods
required to process ancient/historical, and modern genomic data to estimate diversity
Kutschera et al. BMC Bioinformatics (2022) 23:228 Page 15 of 17

changes more streamlined and accessible to a wider conservation community, including

museum curators as well as conservation researchers and practitioners.

Availability and requirements

Project name: GenErode
Project home page: https://​github.​com/​NBISw​eden/​G enEr​ode/
Archived version: 0.4.1
Operating system(s): Linux
Programming language: Snakemake, Python3
Other requirements: Miniconda or Anaconda, Singularity
License: GPL-3.0
Any restrictions to use by non-academics: None.
GenErode is an open-source software and is available on GitHub (https://​github.​com/​
NBISw​eden/​G enEr​ode/), including extensive documentation. GenErode depends on
Snakemake, the Conda package manager and Singularity. Conda can be downloaded
as part of Anaconda or Miniconda (Python 3.7), and Singularity has to be installed.
A Conda environment containing pipeline-wide dependencies (incl. Snakemake) is
available on GitHub (‘environment.yml’). All other dependencies will be automatically
handled by Snakemake using Singularity when running the pipeline.

Abbreviations
CpG Cytosine-guanine dinucleotides
FROH ROH-based inbreeding coefficient
HPC High-performance computing
LOF Loss-of-function
PCA Principal component analysis
PC1 First principal component
ROH Runs of homozygosity
θ Maximum likelihood population mutation parameter (θ = 4Neμ)
UDG Uracil-DNA glycosylase

Supplementary Information
The online version contains supplementary material available at https://​doi.​org/​10.​1186/​s12859-​022-​04757-0.

Additional file 1. Supplementary Information, which contains Extended methods notes 1–2, Tables S1-S3 and
Figures S1–S4
Additional file 2. GenErode pipeline report for analyses of the test dataset based on the white rhinoceros reference.
Additional file 3. GenErode pipeline report for analyses of the test dataset based on the Sumatran rhinoceros
reference.

Acknowledgements
We would like to express our thanks to Pontus Skoglund for providing us with his script ‘samremovedup.py’, Jonas Söder‑
berg for the GenErode pipeline logo design, Camilo Chacón-Duque, Petter Larsson, Heloise Muller, Ioana Meleg, Jovanka
Studerus for testing the pipeline, and Johan Nylander, Marcel Martin, Mahesh Binzer-Panchal, Thijessen Naidoo, Winfried
Kretzschmar, Fredrik Boulund, John Sundh, and Per Unneberg for helpful discussions on the pipeline development.

Author contributions
DDdM, VEK, ND, JvS, BN, and LD conceived the idea. DDdM performed conceptual work. VEK designed and developed
the pipeline. MK and TvV developed parts of the pipeline. DDdM, TvV, ND, DWGS, JvS, EL, MD, BN, and LD provided critical
input for the pipeline development. VEK, ND, JvS, EL, MD, and PE tested the pipeline. VEK generated the test dataset and
analyzed it using the pipeline. DDdM and VEK wrote the manuscript. All authors read and approved the final manuscript.

Funding
Open access funding provided by Stockholm University. DDdM. acknowledges support from the Carl Tryggers Founda‑
tion (CTS 17:109). DWGS. received funding for this project from the European Union’s Horizon 2020 research and innova‑
tion programme under the Marie Skłodowska-Curie grant agreement No 796877. LD acknowledges funding from the
Swedish Research Council (2017-04647) and FORMAS (2015-676). ND acknowledges funding from the Swiss National
Kutschera et al. BMC Bioinformatics (2022) 23:228 Page 16 of 17

Science Foundation (P2SKP3_165031 and P300PA_177845) and the Carl Tryggers Foundation (CTS 19:257). VEK, MK, TvV,
PE and BN are financially supported by the Knut and Alice Wallenberg Foundation as part of the National Bioinformatics
Infrastructure Sweden at SciLifeLab. The computations were enabled by resources provided by the Swedish National
Infrastructure for Computing (SNIC) at UPPMAX, partially funded by the Swedish Research Council through grant agree‑
ment no. 2018-05973. The funders had no role in the design of this study, the collection, analysis, and interpretation of
data or in the writing of the manuscript.

Availability of data and materials

The Sumatran rhinoceros test dataset is available in the Scilifelab Data Repository (https://​doi.​org/​10.​17044/​scili​felab.​
19248​172) and is suitable for testing the GenErode pipeline. A minimum of 2 cores with at least 6 GB each is sufficient for
each of the pipeline jobs to run with this test dataset, so a HPC system is not strictly mandatory. However, the execution
of at least 16 jobs in parallel using the cluster configuration settings in ‘config/cluster.yaml’ from the GitHub repository is
highly recommended to reduce the overall runtime. For test runs of the pipeline with limited computational resources,
a minimal test dataset is available in the ‘.test/data’ folder of the GitHub repository. This data was deposited there for
continuous integration of the pipeline with GitHub actions. Note that the results from this dataset are not biologically
meaningful due to the reduced size of the dataset.

Declarations
Ethics approval and consent to participate
Not applicable.

Consent for publication

Not applicable.

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

Received: 8 March 2022 Accepted: 30 May 2022

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