DOI: 10.5061/dryad.kprr4xhb3
title: Metazoa-level USCOs as markers in species delimitation and classification

description: Metazoa-level Universal Single-Copy Orthologs (USCOs) are universally
 applicable markers for DNA taxonomy in animals which can replace or
 supplement single-gene barcoding. While Metazoa-level USCOs from target
 enrichment data were shown to reliably distinguish species, it remains to
 be tested whether USCOs are an evenly distributed, representative sample
 of a given metazoan genome, and hence can facilitate detection of past
 hybridization events. Besides, unlinked loci are a principal assumption in
 coalescent-based species delimitation approaches. 239 chromosome-level
 genomes were analyzed to show that Metazoa-level USCOs are a
 representative sample of a genome: in terms of distances to each other on
 a chromosome, but also over the chromosomes, they are almost as evenly
 distributed as protein-coding genes in general are. We tested the
 suitability of Metazoa-level USCOs extracted from genomes for species
 delimitation and phylogeny in four case studies: Anopheles mosquitos,
 Drosophila fruit flies, Heliconius butterflies, and Darwin’s
 finches.  In almost all instances USCOs allowed delineating
 species and yielded phylogenies that correspond to those generated from
 whole genome data. Our results show that USCO genes can be considered as
 genetically unlinked for practical purposes and representative for an
 entire metazoan genome. Our phylogenetic analyses demonstrate that USCOs
 may complement single-gene barcoding and provide more accurate taxonomic
 inferences. Combining USCOs from sources that used different versions of
 ortholog reference libraries to infer marker orthology may be challenging
 and at times impact taxonomic conclusions. However, we expect this problem
 to become less severe as the size of genome reference libraries and their
 sampling of organismic lineages is rapidly increasing.
description: Supplementary files for study "From benchmarking to systematics:
 exploiting metazoan USCOs from whole genome data", L. Dietz et al.,
 submitted For questions, contact Dirk Ahrens
 () or Lars Dietz
 () Each of these ZIP files contains a folder
 with several files containing alignments, phylogenetic trees, or other
 data. For more information on these files, please consult the accompanying
 description files. All alignments, including SNP datasets, are in FASTA
 format and can be opened with standard alignment viewers. Except in the
 metazoan alignments, ambiguity codes (R, Y, W, S, M, K) stand for
 positions inferred to be heterozygous. Phylogenetic trees are in NEWICK
 format and can be opened in a standard phylogenetic tree viewer such as
 FigTree. All trees are unrooted. Partition files for concatenated
 alignments are in NEXUS format for use in IQ-TREE. SNP datasets recoded
 for NMDS analysis are tab-delimited text and numbers have the following
 meaning: 0: homozygous for more common allele, 1: heterozygous, 2:
 homozygous for less common allele. Unknown positions are represented by
 empty cells. Histograms of distances between USCOs are in PDF format.
 Other figures are in SVG format and can be opened e.g. with Inkscape.
 Histograms of USCO All other files can be opened with a standard text
 editor. ## Description of the data and file structure
 all_data_astral_trees.zip: Coalescent-based trees from ASTRAL analysis of
 gene trees based on USCO DNA sequences extracted with all three
 approaches. (see all_data_astral_trees_descriptions.txt for details)
 all_data_concat_alignments.zip: Concatenated alignments of USCO DNA
 sequences extracted with all three approaches. (see
 all_data_concat_alignments_descriptions.txt for details)
 all_data_concat_trees.zip: Phylogenetic trees based on concatenated
 alignments of USCO DNA sequences extracted with all three approaches. (see
 all_data_concat_trees_descriptions.txt for details)
 all_data_gene_alignments.zip: DNA alignments of individual USCO loci
 extracted with all three approaches. (see
 all_data_gene_alignments_descriptions.txt for details)
 all_data_gene_trees.zip: Phylogenetic trees based on DNA alignments of
 individual USCO loci extracted with all three approaches. (see
 all_data_gene_trees_descriptions.txt for details)
 all_data_partition_files.zip: Partition files for concatenated alignments
 of USCO DNA sequences extracted with all three approaches. (see
 all_data_partitions_descriptions.txt for details) astral_trees.zip:
 Coalescent-based trees from ASTRAL analysis of gene trees based on DNA
 alignments of individual USCO loci of the four study cases.
 concat_alignments.zip: Concatenated alignments of USCO DNA sequences of
 the four study cases. (see concat_alignments_descriptions.txt for details)
 concat_trees.zip: Phylogenetic trees based on concatenated alignments of
 USCO DNA sequences of the four study cases. (see
 concat_alignments_descriptions.txt for details) gene_alignments.zip: DNA
 alignments of individual USCO loci of the four study cases. (see
 gene_alignments_descriptions.txt for details) gene_trees.zip: Phylogenetic
 trees of individual USCO loci of the four study cases. (see
 gene_trees_descriptions.txt for details) metazoa_busco_aa_alignments.zip:
 Alignments of protein sequences of individual USCO genes extracted from
 metazoan genomes with BUSCO, with badly aligned regions removed.
 metazoa_busco_aa_trees.zip: Phylogenetic trees based on protein sequences
 of individual USCO genes extracted from metazoan genomes with BUSCO.
 metazoa_busco_nt_alignments.zip: Alignments of DNA sequences (1st and 2nd
 codon positions) of individual USCO genes extracted from metazoan genomes
 with BUSCO, with badly aligned regions removed.
 metazoa_busco_nt_trees.zip: Phylogenetic trees based on DNA sequences (1st
 and 2nd codon positions) of individual USCO genes extracted from metazoan
 genomes with BUSCO. nmds_plots.zip: NMDS plots based on SNPs from USCO
 loci of the four study cases. (see nmds_plots_descriptions.txt for
 details) partition_files.zip: Partition files for concatenated alignments
 of USCO DNA sequences of the four study cases. (see
 partition_files_descriptions.txt for details) snp_alignments.zip:
 Alignments of informative SNPs from USCO loci of the four study cases.
 (see snp_alignments_descriptions.txt for details) snp_nmds.zip: Tables of
 biallelic SNPs for NMDS analysis from USCO loci of the four study cases.
 (see snp_nmds_descriptions.txt for details) species_delimitation.zip:
 Tables of species-level entities inferred with SODA and tr2 based on USCO
 loci of the four study cases. (see species_delimitation_descriptions.txt
 for details) structure_plots.zip: Results of STRUCTURE clustering based on
 SNPs from USCO loci of the four study cases. (see
 structure_plots_descriptions.txt for details) usco_position_diffs.zip:
 Tables in tsv format containing distances between starting points of
 neighboring USCO genes in metazoan genomes. Columns are, from left to
 right, OrthoDB ID numbers of the genes, distance between them, logarithm
 of the distance, distance normalized to genome size, and logarithm of the
 distance normalized to genome size. usco_position_tables.zip: Tables in
 tsv format output by BUSCO containing positions of USCO genes in metazoan
 genomes. Columns are, from left to right, OrthoDB ID number of the gene,
 status, NCBI accession number of the chromosome/contig sequence, starting
 position of the coding sequence, end position, quality score, and length.
 uscos_per_chromosome.zip: Tables showing, for each analyzed genome, the
 name of each chromosome, its length in base pairs, the number of all
 protein-coding genes it contains, and number of mzl-USCOs it contains.
 metazoa_busco_aa_astral.tre: Coalescent-based tree from ASTRAL analysis of
 gene trees based on protein alignments of USCO genes in metazoan genomes.
 metazoa_busco_aa_concat.tre: Maximum-likelihood tree based on concatenated
 protein alignments of USCO genes in metazoan genomes.
 metazoa_busco_nt12_astral.tre: Coalescent-based tree from ASTRAL analysis
 of gene trees based on DNA alignments (1st and 2nd codon positions) of
 USCO genes in metazoan genomes. metazoa_busco_nt12_concat.tre:
 Maximum-likelihood tree based on concatenated DNA alignments (1st and 2nd
 codon positions) of USCO genes in metazoan genomes.
 usco_co-occurrence.txt: Table of frequencies with which pairs of USCO
 genes occur on the same chromosome across metazoan taxa. Columns are, from
 left to right, OrthoDB ID number of first gene, OrthoDB ID number of
 second gene, and frequency. usco_diff_histograms.pdf: Histograms of
 distances between starting points of neighboring USCO genes in metazoan
 genomes. x-axis shows logarithm of distance, y-axis shows number of
 distances in the respective size class. usco_diff_histograms_normed.pdf:
 Histograms of distances between starting points of neighboring USCO genes
 in metazoan genomes normalized by genome size. x-axis shows logarithm of
 normalized distance, y-axis shows number of distances in the respective
 size class. ## Sharing/Access information Links to other publicly
 accessible locations of the data: * none Data was derived from the
 following sources: * Metazoan genome assemblies at NCBI
 (), see list in S1 of paper * Raw
 reads from genome sequencing projects of Anopheles, Drosophila, Heliconius
 and Darwin's finches at NCBI (),
 see list in S2 of paper ## Code/Software removegaps_d.pl: This script
 removes all positions from FASTA alignments for which sequence data is
 available in less than a specified number of taxa. Requires, in that
 order, the path to the folder containing input alignments, the minimum
 number of taxa for which sequence information must be available for
 alignment positions to be kept, and a folder where output alignments are
 put. Input alignment file names must end in .fas. Example: removegaps_d.pl
 input_folder/ 5 output_folder/ removegaps_snp_inf_d.pl: This script
 removes non-parsimony-informative SNPs from SNP datasets in FASTA format.
 Requires, in that order, the path to the folder containing input data and
 a folder where output alignments are put. Input file names must end in
 .fas. Example: removegaps_snp_inf_d.pl input_folder/ output_folder/
 removegaps_snp_d.pl: This script removes all positions from SNP datasets
 in FASTA format that are missing in more than a specified number of taxa.
 It is recommended to initially set this to 0 or some other small number,
 then delete all empty files from your output folder, and increase by one
 and repeat until no more empty files are produced. Input file names must
 end in .fas. Requires, in that order, the path to the folder containing
 input alignments, the maximum number of taxa containing a gap, and a
 folder where output alignments are put. Example: removegaps_snp_d.pl
 input_folder/ 0 output_folder/ snp-pca_d.pl: This script converts an SNP
 dataset in FASTA format, including only SNPs with at most two alleles, to
 a format usable for PCA or NMDS. A majority consensus sequence of the
 input alignment, as a file containing only the sequence, must be created
 beforehand. Requires, in that order, name of the input, consensus, and
 output files. Input alignment file names must end in .fas. Example:
 snp-pca_d.pl input.fas cons.txt output.txt extract_codpos_d.pl: This
 script removes the third codon position from all nucleotide alignments
 (FASTA format) in a folder. Requires, in that order, the paths to the
 folder containing input alignments and an output folder for modified
 alignments. Names of alignment files must end in .fas. Example:
 extract_codpos_d.pl input_folder/ output_folder/ concat_eogs_part_d.pl:
 This script creates a concatenated alignment FASTA file from all
 alignments (FASTA format) in a folder. It also creates a partition file in
 NEXUS format listing each alignment as a partition. Requires, in that
 order, the path to the folder containing input alignments, a name for the
 concatenated output alignment, and the partition file. Names of alignment
 files must end in .fas. Example: concat_eogs_part_d.pl input_folder/
 concat.fas partition.nex extract_chromo_d.pl: This script removes all
 non-chromosome contigs from eukaryotic genomes in FASTA format downloaded
 from NCBI. Requires, in that order, an input folder containing genome
 files and an output folder for genome files containing only chromosomes.
 Genome files must end in .fna. Example: extract_chromo_d.pl input_folder/
 output_folder/ genomesize_d.pl: This script creates a table of genome
 sizes in base pairs from genome files in FASTA format. Requires, in that
 order, an input folder containing genome files and a name for the genome
 size table. Genome files must end in .fna. Example: genomesize_d.pl
 input_folder/ genomesize.txt busco_diff_d.pl: This script creates tables
 of absolute and normalized distances and their logarithms between
 neighboring USCO genes in metazoan genomes based on a BUSCO output table.
 Requires, in that order, a table of genome sizes created with
 genomesize_d.pl, an input folder containing the BUSCO output tables for
 each genome, and an output folder for the tables. Genome files must be in
 FASTA format and end in .fna. Names of BUSCO output tables must be of the
 form taxon.tsv, where “taxon” is the taxon name listed in the genome size
 table. Example: busco_diff_d.pl genomesize.txt input_folder/
 output_folder/ busco_diff_freq_d.pl: This script creates a table of
 frequencies of normalized logarithmic size classes of distances between
 neighboring USCO genes in metazoan genomes. Requires, in that order, an
 input folder containing a table of distances created with busco_diff_d.pl,
 and a name for the output file. Example: busco_diff_freq_d.pl
 input_folder/ sizefreqs.txt busco_diff_median_d.pl: This script creates a
 table of medians of distances between neighboring USCO genes in metazoan
 genomes. Requires, in that order, an input folder containing a table of
 distances created with busco_diff_d.pl, and a name for the output file.
 Example: busco_diff_freq_d.pl input_folder/ usco_medians.txt
 gene_random_diffs_d.pl: This script creates tables containing the median
 distance between neighboring genes from a random sample of genes from a
 set of genomes. CDS files of each genome must be in the folder structure
 as downloaded from NCBI. Requires, in that order, a tab-separated table
 containing the accession number for each genome and the number of genes to
 be sampled from the genome, a folder containing all sub-folders with the
 CDS files of each genome, the number of times the sampling is to be
 repeated, and an output folder for the files containing median distances
 for each genome. Example: gene_random_diffs_d.pl cds_files/ 10000
 output_folder/ gene_sample_median_d.pl: This script creates a table
 containing, in that order, the median distance between neighboring
 randomly sampled genes, the quotient of the distance between neighboring
 USCO genes by that number, and the proportion of random samples for which
 the median distance of randomly sampled genes is lower than that of USCO
 genes. Requires, in that order, the directory with the tables output by
 gene_random_diffs_d.pl, the table output by busco_diff_median_d.pl, the
 number of times the sampling was repeated, and a name for the output file.
 Example: gene_sample_median_d.pl median_tables/ usco_medians.txt 10000
 median_compare.txt usco_chromo_numbers_d.pl: This script creates, for each
 of a set of genomes, a table containing, in that order, the names of all
 chromosomes, their lengths in base pairs, the number of protein coding
 genes in each chromosome, and the number of complete single-copy USCO
 genes in it. Requires, in that order, a tab-separated table containing the
 accession numbers of all genomes and the name of the respective organism,
 a folder containing sub-folders with the sequence reports from NCBI for
 all genomes, a folder containing sub-folders with the CDS files, a folder
 containing the tables output by BUSCO listing all USCOs found in the
 genome, and a folder for the output files. Sequence reports and CDS files
 of each genome must be in the folder structure as downloaded from NCBI.
 Names of BUSCO output tables must contain the organism name (e.g.
 Homo_sapiens.tsv). Example: usco_chromo_numbers_d.pl genome_table.txt
 sequence_reports/ cds_files/ busco_tables/ output_folder/
 usco_evenness_d.pl: This script creates a table listing, for each of a set
 of genomes, the evenness (defined as e^H/S, with H being the entropy of
 the distribution and S being number of chromosomes) of, in that order,
 chromosome length, number of protein-coding genes per chromosome, and
 number of USCO genes per chromosome. Requires, in that order, the folder
 containing tables output by usco_chromo_numbers_d.pl, and a name for the
 output file. Example: usco_evenness_d.pl chromo_numbers/ evenness.txt
 usco_chi_square_d.pl: This script creates a table listing, for each of a
 set of genomes, the chi-square value showing the deviation of the actual
 distribution of USCOs between chromosomes from a distribution proportional
 to chromosome sizes, the chi-square value showing the deviation from a
 distribution proportional to the number of protein-coding genes in
 general, and the number of degrees of freedom. p-values can then be
 calculated e.g. by Microsoft Excel. Requires, in that order, the folder
 containing tables output by usco_chromo_numbers_d.pl, and a name for the
 output file. Example: usco_chi_square_d.pl chromo_numbers/ chi_square.txt
 busco_chromo_table_d.pl: This script creates a table of proportions with
 which any pairs of USCO genes occur on the same chromosome across a number
 of genomes. Requires, in that order, an input folder containing BUSCO
 output files and a name for the output table. Names of input files must
 end in .tsv. Example: busco_chromo_table_d.pl input_folder/
 chromo_table.txt usco_overlap_d.pl: This script produces an upper diagonal
 matrix with the proportion of overlapping positions between each pair of
 taxa in an alignment. Input alignment must be in FASTA format, with each
 sequence in a single line. Requires, in that order, name of a text file
 listing the taxa in correct order, the input alignment, and the output
 file. Example: usco_overlap_d.pl taxonlist.txt input.fas output.txt