DOI: 10.5061/dryad.612jm64cr
title: Validating a target-enrichment design for capturing uniparental haplotypes in ancient domesticated animals

description: In the last three decades, DNA sequencing of ancient animal osteological
 assemblages has become an important tool complementing standard
 archaeozoological approaches to reconstruct the history of animal
 domestication. However, osteological assemblages of key archaeological
 contexts are not always available or do not necessarily preserve
 sufficient amounts of ancient DNA for a cost-effective genetic analysis.
 Here, we develop an in-solution target-enrichment approach, based on
 80-mer species-specific RNA probes (ranging from 306 to 1,686 per species)
 to characterize (in single experiments) the mitochondrial genetic
 variation from eight domesticated animal species of major economic
 interest: cattle, chickens, dogs, donkeys, goats, horses, pigs, and sheep.
 We also illustrate how our design can be adapted to enrich DNA library
 content and map the Y-chromosomal diversity within Equus caballus. By
 applying our target-enrichment assay to an extensive panel of ancient
 osteological remains, farm soil, and cave sediments spanning the last 43
 kyrs, we demonstrate that minimal sequencing efforts are necessary to
 exhaust the DNA library complexity and to characterize mitogenomes to an
 average depth-of-coverage of 19.4 to 2,003.7 -fold. Our assay further
 retrieved horse mitogenome and Y-chromosome data from Late Pleistocene
 coprolites, as well as bona fide mitochondrial sequences from species that
 were not part of the probe design, such as bison and cave hyena. Our
 methodology will prove especially useful to minimize costs related to the
 genetic analyses of maternal and paternal lineages of a wide range of
 domesticated and wild animal species, and for mapping their diversity
 change over space and time, including from environmental samples. Here we
 provide the fasta files of the probes. 
description: We screened the literature to collect an extensive representation
 of full mitogenome sequences from a total of eight species of domesticated
 animals, namely cattle (<em>Bos taurus</em>), chickens
 (<em>Gallus gallus</em>), dogs (<em>Canis
 familiaris</em>), donkeys (<em>Equus asinus</em>), goats
 (<em>Capra aegagrus hircus</em>), horses (<em>Equus
 caballus</em>), pigs (<em>Sus scrofa domesticus</em>),
 and sheep (<em>Ovis aries</em>) (Table S2). We then added the
 mitochondrial haplotypes from three equid and three canid outgroups
 (<em>Equus hemionus</em>, N=1; <em>E, africanus
 somaliensis</em>, N=1, and <em>E</em><em>.
 hydruntinus</em>, N=1; <em>Can</em><em>is
 latrans</em>, N=5; <em>Canis himalayensis</em>, N=2, and
 <em>C</em><em>. lupus signatus</em>, N=2,
 respectively) to the <em>E. asinus</em> and
 <em>C</em><em>. familiaris </em>data sets. We also
 added to this dataset 476 full mitogenome sequences from hominins,
 including 433 <em>Homo sapiens</em>
 <em>sapiens</em> with a worldwide distribution, 34
 <em>Homo sapiens neanderthaliensis</em>, seven <em>Homo
 altaiensis </em>and two <em>Homo heidelbergensis</em>.
 We also included a total of 409 bear mitochondrial sequences.
 Independent multiple sequence alignments (MSA) were
 built for each of species above emntioned using MAFFT v7.453 (Nakamura et
 al. 2018), and manually curated with AliView v1.28 (Larsson
 2014) and Seaview v4.7 (Gouy, Guindon
 & Gascuel 2010). A
 maximum likelihood (ML) tree was then generated using IQ-Tree v2.0.3
 (Minh et al. 2020), with 1,000 bootstrap
 pseudoreplicates (option -b 1000) for each data set and visualized in iTOL
 v6.7 (Letunic & Bork 2021). The resulting tree topologies were
 manually compared with those published to confirm the haplogroup structure
 within each species group. MSAs were used to identify those biallelic
 Single Nucleotide Polymorphisms (SNPs) private to each individual
 haplogroup, which were used to prioritize probe design. In a first step,
 we defined a set of 90 bp long primary probes centered on every SNP
 defining a haplogroup, considering as candidate probes all reported
 haplotypes starting 45 and 44 nucleotides prior to the underlying SNP
 position. This first step was necessary to account for the possible
 presence of deletions in specific MSA regions. DNA variation consisting of
 gaps larger than three nucleotides and/or present only once were
 disregarded. In a second step, we defined a set of 80-mer candidate probes
 by further trimming each individual 90-mer symmetrically from both ends.
 Those probes showing %GC values within the 0.33 to 0.6 range were
 retained, and in the case where two or more probes were available for the
 target SNP, the one showing the highest entropy value, indicative of
 greater information content, hence, sequence complexity, was
 preferentially selected (otherwise, the selection was random), delivering
 the final set of 80-mer probes (Fig. 1). Our procedure resulted in a final
 set of 306 to 1,686 probes per species group (Table S2). The sites
 overlapping with probes in each MSA were sub-selected for ML tree
 reconstruction in IQ-Tree, following the same procedure as above, to
 confirm their capacity to accurately identify the entire range of DNA
 variation. A total of 18,780 probes targeting Y-chromosomal variation in
 horses were designed by applying the same procedure to an MSA containing
 403 horse Y-chromosomal sequences (272 ancient and 131 modern; Fages et
 al. 2019, Librado et al. 2021, 2024), with the exception that 60-mers were
 considered instead of 80-mers. The mitochondrial and Y-chromosomal probe
 sequences were then sent to Arbor Biosciences, Ann Arbor, USA for
 production as two independent sets of RNA baits. These sets are provided
 as Supplementary File X and Supplementary File Y.
description: # Validating a target-enrichment design for capturing uniparental
 haplotypes in ancient domesticated animals
 [https://doi.org/10.5061/dryad.612jm64cr](https://doi.org/10.5061/dryad.612jm64cr) ## Description of the data and file structure We screened the literature to collect an extensive representation of full mitogenome sequences from a total of eight species of domesticated animals, namely cattle (*Bos taurus*), chickens (*Gallus gallus*), dogs (*Canis familiaris*), donkeys (*Equus asinus*), goats (*Capra aegagrus hircus*), horses (*Equus caballus*), pigs (*Sus scrofa domesticus*), and sheep (*Ovis aries*) (Table S2). We then added the mitochondrial haplotypes from three equid and three canid outgroups (*Equus hemionus*, N=1; *E, africanus somaliensis*, N=1, and *E. hydruntinus*, N=1; *Canis latrans*, N=5; *Canis himalayensis*, N=2, and *C. lupus signatus*, N=2, respectively) to the *E. asinus* and *C. familiaris *data sets. We also added to this dataset 476 full mitogenome sequences from hominins, including 433 *Homo sapiens* *sapiens* with a worldwide distribution, 34 *Homo sapiens neanderthaliensis*, seven *Homo altaiensis *and two *Homo heidelbergensis*. We also included a total of 409 bear mitochondrial sequences. Independent multiple sequence alignments (MSA) were built for each of species above emntioned using MAFFT v7.453 (Nakamura et al. 2018), and manually curated with AliView v1.28 (Larsson 2014) and Seaview v4.7 (Gouy, Guindon & Gascuel 2010). A maximum likelihood (ML) tree was then generated using IQ-Tree v2.0.3 (Minh et al. 2020), with 1,000 bootstrap pseudoreplicates (option -b 1000) for each data set and visualized in iTOL v6.7 (Letunic & Bork 2021). The resulting tree topologies were manually compared with those published to confirm the haplogroup structure within each species group. MSAs were used to identify those biallelic Single Nucleotide Polymorphisms (SNPs) private to each individual haplogroup, which were used to prioritize probe design. In a first step, we defined a set of 90 bp long primary probes centered on every SNP defining a haplogroup, considering as candidate probes all reported haplotypes starting 45 and 44 nucleotides prior to the underlying SNP position. This first step was necessary to account for the possible presence of deletions in specific MSA regions. DNA variation consisting of gaps larger than three nucleotides and/or present only once were disregarded. In a second step, we defined a set of 80-mer candidate probes by further trimming each individual 90-mer symmetrically from both ends. Those probes showing %GC values within the 0.33 to 0.6 range were retained, and in the case where two or more probes were available for the target SNP, the one showing the highest entropy value, indicative of greater information content, hence, sequence complexity, was preferentially selected (otherwise, the selection was random), delivering the final set of 80-mer probes (Fig. 1). Our procedure resulted in a final set of 306 to 1,686 probes per species group (Table S2). The sites overlapping with probes in each MSA were sub-selected for ML tree reconstruction in IQ-Tree, following the same procedure as above, to confirm their capacity to accurately identify the entire range of DNA variation. A total of 18,780 probes targeting Y-chromosomal variation in horses were designed by applying the same procedure to an MSA containing 403 horse Y-chromosomal sequences (272 ancient and 131 modern; Fages et al. 2019, Librado et al. 2021, 2024), with the exception that 60-mers were considered instead of 80-mers. The mitochondrial and Y-chromosomal probe sequences were then sent to Arbor Biosciences, Ann Arbor, USA for production as two independent sets of RNA baits. These sets are provided as Supplementary File X and Supplementary File Y. ### Files and variables #### File: Supplementary\_File\_Y.zip **Description:**  This file contains fasta sequences of Y chromosomal probes.  #### File: Supplementary\_File\_X.zip **Description:** This file contains fasta sequences of Mitochondrial probes. Species name are written in the fasta heading of each probe sequence  while the number denotes the position of target SNP of that probe when compared reference sequence.