Validating a target-enrichment design for capturing uniparental haplotypes in ancient domesticated animals

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.

在过去三十年中，针对古代动物骨骼遗存组合的DNA测序已成为辅助标准动物考古学（archaeozoological）研究方法、重构动物驯化历史的重要工具。然而，关键考古背景下的骨骼遗存组合并非总能获取，也未必能保存足量的古代DNA以开展经济高效的遗传分析。本研究开发了一种基于80聚体（80-mer）物种特异性RNA探针（RNA probes，单物种探针数量介于306至1686个区间）的液相靶向富集（in-solution target-enrichment）方法，可在单次实验中表征八大经济价值突出的家养动物物种的线粒体遗传变异：牛、鸡、犬、驴、山羊、马、猪与绵羊。我们还展示了该探针设计方案可如何被改造，以富集DNA文库序列，并绘制马（Equus caballus）的Y染色体多样性图谱。通过将本靶向富集检测方法应用于覆盖过去4.3万年的海量古代骨骼遗存、农田土壤与洞穴沉积物样本，我们证实仅需极低的测序工作量即可充分解析DNA文库的序列复杂性，并将线粒体基因组（mitogenome）的平均测序覆盖深度达到19.4倍至2003.7倍。本检测方法还从晚更新世粪化石（coprolites）中获取了马的线粒体基因组与Y染色体数据，同时从探针设计未覆盖的物种（如野牛（bison）与洞鬣狗（cave hyena））中获取了真实可靠的线粒体序列。该方法将尤其有助于降低针对大量家养与野生动物物种的母系、父系谱系开展遗传分析的相关成本，并可用于绘制这些物种的多样性随空间与时间的变化规律，包括来自环境样本的材料。本研究附带提供了探针的FASTA格式文件。