- inference of kinship, ancestry and disease susceptibility in orang-utans (genus: Pongo). - Background: Orang-utans comprise three critically endangered species endemic to the islands of Borneo and Sumatra. - Results: We identified and present genomic co-ordinates for 175,186 SNPs and 2315 Y-chromosomal targets, plus 185 genes either known or presumed to be pathogenic in cardiovascular (N = 109) or respiratory (N = 43) diseases in humans – the primary and secondary causes of captive orang-utan mortality – or a majority of other human diseases (N = 33). - Conclusions: Our targets are of broad utility in studies of orang-utan ancestry, admixture and disease susceptibility and aetiology, and thus are of value in addressing questions key to the survival of these species. - To facilitate comparative analyses, these targets could now be standardized for future orang-utan population genomic studies.. - Advances in analytic molecular methods have gradually shed light on the evolutionary history of orang-utans (Pongo spp. - Protein electrophoretic studies, beginning in the 1970s [1, 2], first supported the description of two subspecies, distinct to the islands of Borneo and. - The first orang-utan reference genome was generated in 2011 [6], before the genus was split into three species in 2017, following whole genome re-sequencing of a previ- ously understudied population [7]. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - latter is still divided into three subspecies in the western (P. - morio) regions of the island [4, 5].. - Our understanding of orang-utan taxonomy and evo- lution has fast outpaced their survival. - More than 100, 000 Bornean orang-utans were reportedly killed in the wild from 1999 to 2015, 50% of which were lost from forests affected by natural resource extraction [8]. - 57,000 reportedly survive on Borneo, while ~ 13,800 Sumatran and ~ 800 Tapanuli orang-utans are thought to remain on Sumatra [9]. - Consequently, surviving wild orang-utans are increasingly intensively managed by humans, whether intended or not. - Long runs of homozy- gosity have been observed in the genomes of wild Tapa- nuli orang-utans, suggesting inbreeding is occurring due to anthropogenic range restriction [7]. - On Borneo, orang-utans of non-native subspecies are known to have been translocated and unwittingly returned to the wild, despite diverging ~ 176,000 years ago, and being subject to marked genetic differentiation over the last ~ 82,000 years [10]. - 1500 orang-utans are still awaiting reintroduction from rehabilitation centres in- situ. - Though the potential for outbreeding depression has been cited, orang-utans’ large home ranges and long generation times render it impractical to investigate its incidence in the wild [11].. - In contrast, ex-situ orang-utans in zoos might serve as model populations for studying the effects of human intervention. - Approximately 1100 orang-utans live in zoos worldwide, although numbers are probably higher in developing nations and in range countries [12]. - Zoo populations of orang-utans are known to be highly admixed. - Until the 1990s, Bornean and Sumatran orang- utans were inter-bred in zoos, producing a hybrid popu- lation that has since been contracepted. - Beyond the species level, captive Sumatran orang- utans have been shown to be highly admixed among those from distinct geographic subpopulations, while those of Bornean origin are known to have introgressed among all three subspecies. - These hybridizations have occurred rapidly over multiple generations, given the far shorter inter-birth intervals than would naturally occur in the wild [13]. - As we consider how best to manage displaced orang- utans [11, 18], and how best to secure a sustainable fu- ture for those in zoos (sensu [19. - These studies lack the resolutions necessary to build distant pedigrees, however, and – as so many orang-utans are now unnaturally admixed, both in ex-situ and reintro- duced populations – their methods use too few loci to infer complex hybridization [30]. - Here, we present a panel of molecular targets that can facilitate standardized comparative studies of orang-utan genomic variation. - These markers are of broad utility in studies that seek to better under- stand orang-utan evolutionary biology and health.. - Selection of ancestry- and kinship-informative SNPs We mapped published sequence reads from 37 whole genomes, derived from three prior studies to the latest iteration of the orang-utan reference genome (ponAbe3, [32]) (Table 1). - To discover low-frequency alleles across the genus, we ap- plied the workflow four times: first, comprising all ge- nomes, and subsequently, comprising genomes from each orang-utan species separately. - Capitalizing on the new –include-non-variant-sites flag in the GATK 4.1.2.0, we then re-called haplotypes and re-genotyped all samples, using the master loci set as an interval list. - We supplemented these with 51,128 additional SNP positions derived from 71 zoo-housed orang-utans that we genotyped from whole blood or tissue-derived DNAs on the Illumina iScan platform. - We first extracted gen- omic DNA using either the Maxwell RSC Blood DNA or Tissue DNA kits, respectively, as automated on the Table 1 Published, re-sequenced genomes from 37 orang-utans. - 0.01 and converted the final GenomeStudio file to VCF using the iScanVCFMerge tool (Fountain et al., in review).. - In the absence of a Y chromosome in the (female) orang-utan reference genome (ponAbe3), we designed probes for human (hg19) SNP positions that can be con- sistently successfully target-enriched in commercial hu- man SeqCap panels. - As numerous prior studies have successfully mapped male orang-utan sequences to the human Y-chromosome, we anticipated high on-target hybrid capture efficiency [31].. - First, through a literature review, we prepared a list of genes either known or presumed to be pathogenic for cardio- vascular and/or chronic respiratory diseases in humans, capitalizing on the genetic similarity of the human and orang-utan genomes. - This process enabled us to view and search for documented orthologs within the orang-utan genome, and to determine their start and end positions. - For any genes in these panels not on our prior list, we principally used the UCSC Table Browser to derive exon positions for each gene on the orang-utan genome. - For those not present in the Table Browser, we retrieved exon posi- tions from the annotated Generic Feature Format (.GFF) file.. - These might therefore be linked to health disorders or be indi- cators of in- and outbreeding depression in orang-utans.. - To evaluate the utility of SeqCap technology in orang- utans, we first applied the SeqCap EZ MedExome panel – designed to target enrich the human exome, with higher coverage of medically relevant genes – to gen- omic DNA derived from nine orang-utans. - We used the resulting sequence data as a reference when designing (or re-designing) probes around our custom orang-utan targets.. - To prevent cross- hybridization to untargeted loci, we removed any probes containing 15-mers overrepresented in the ponAbe3 build. - We present ponAbe3 genomic co-ordinates for 175,186 SNP loci, of which 124,060 were derived from our GATK analysis of published orang-utan whole-genome. - sequences and 51,126 from novel iScan genotyping of orang-utans. - where applicable) are re- ported in the supporting document (SNP_Targets_. - plus all 33 of the additional genes from the ACMG SF v2.0. - and for the study of genes potentially pathogenic for disease in orang-utans. - In the longer term, our panel could be expanded to include other valuable targets. - in orang-utans have shown especially diverse and complicated MHC transcription profiles. - More focused studies of the orang-utan MHC are thus needed to better define the target, in order to fa- cilitate effective probe design. - ‘backwards compatibility’ with the volumes of micro- satellite genotype data generated in the genus to date.. - Developing technologies now render this achievable, even with the highly degraded and non- invasively produced samples that constitute the ma- jority of orang-utan DNA collected to date: notably, fluorescence-activated cell-sorting (fecalFACS) has fa- cilitated high-coverage, minimally biased sequencing of an entire mammalian genome from faeces [53].. - At present, the feasibility of Seq- Cap with orang-utan targets is comparable to what can be achieved using off-the-shelf human-target- enrichment products, in that certain regions present technical challenges in both species. - A prominent sec- tion of the orang-utan BRCA1 gene, for example, comprises a single repeat and corresponds to the same section of human BRCA1 that is similarly diffi- cult to sequence and not often covered by human medical exome kits. - Data are presented for all those loci in the panel, and again for only those loci for which SeqCap probes could be successfully designed. - Further statistics can be found in the supplementary data (SNP_Targets_ponAbe3_bed_file.txt). - In the case of SeqCap technology, dual- ver- sus single-indexing can be used to increase multiplex- ing capacity, maintaining high sequencing coverage while avoiding excessive amounts of data from small target sizes [54]. - This panel has now been standardized for use in The Orang-utan Conservation Genetics Project, a global ef- fort to study the genetics of wild, ex-captive and zoo- housed orang-utans. - More than 3200 DNA samples have been collected globally from orang-utans to date.. - Using the SeqCap technology described herein, we are enriching and sequencing this panel of targets in ~ 1000 individual orang-utans. - We encourage other re- searchers to adopt this panel to facilitate comparative studies of orang-utan population genomics. - List of medically relevant genes in the panel.. - We thank all contributing zoos, plus the Orangutan Species Survival Plan (SSP) for providing approval by recommendation to its member institutions in the US. - GLB thanks Jon Levine, Deb Jurmu and the Wisconsin National Primate Research Center for housing The Orang-utan Conservation Genetics Project, plus all of the Project ’ s prior host institutions: the University of Cambridge, U.K.. - GLB directs The Orang-utan Conservation Genetics Project in the Wisconsin National Primate Research Center at the University of Wisconsin – Madison;. - and The Orang-utan Conservation Genetics Trust. - now The Orang-utan Conservation Genetics Project, Inc. - 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