Pulling out the 1%: Whole-Genome In-Solution (WISC) capture for the targeted enrichment of ancient DNA sequencing libraries. C. D. Bustamante1, M. L. Carpenter1, J. D. Buenrostro1, C. Valdiosera2,3, H. Schroeder2, M. E. Allentoft2, M. Sikora1, M. Rasmussen2, S. Guillén4, G. Nekhrizov5, K. Leshkatov6, D. Dimitrova5, N. Theodossiev6, D. Pettener7, D. Luiselli7, A. E. Moreno1, S. Gravel8, Y. Li8, J. Wang8,9,10,11, M. T. P. Gilbert2,12, E. Willerslev2, W. J. Greenleaf1 1) Genetics, Stanford University School of Medicine, Stanford, CA; 2) Natural History Museum of Denmark, Centre for GeoGenetics, Copenhagen, DK-1350 Denmark; 3) Department of Archaeology, Environment, and Community Planning, Faculty of Humanities and Social Sciences, La Trobe University, Melbourne, VIC 3086, Melbourne, Australia; 4) Centro Mallqui, Calle Ugarte y Moscoso 165, San Isidro, Lima 27, Peru; 5) Bulgarian Academy of Sciences, National Institute of Archaeology, Sofia, 1000, Bulgaria; 6) Department of Archaeology, Sofia University, Sofia, 1504, Bulgaria; 7) Department of Biological, Geological, and Environmental Sciences, University of Bologna, Bologna, Italy; 8) BGI-Shenzhen, Shenzhen 518083, China; 9) King Abdulaziz University, Jeddah, Saudi Arabia; 10) Department of Biology, University of Copenhagen, Copenhagen, Denmark; 11) Macau University of Science and Technology, Macau, China; 12) Ancient DNA Laboratory, Murdoch University, South St, 6150 Western Australia, Australia.

   The very low levels of endogenous DNA remaining in most ancient specimens has precluded the shotgun sequencing of many interesting samples due to cost. For example, ancient DNA (aDNA) libraries derived from bones and teeth often contain <1% endogenous DNA, meaning that the majority of sequencing capacity is taken up by environmental DNA. We will present a method for the targeted enrichment of the endogenous component of human aDNA sequencing libraries. Using biotinylated RNA baits transcribed from genomic DNA libraries, we are able to significantly enrich for human-derived DNA fragments. This approach, which we call whole-genome in-solution capture (WISC), allows us to obtain genome-wide ancestral information from ancient samples with very low endogenous DNA contents. We demonstrate WISC on libraries created from four Iron Age and Bronze Age human teeth from Bulgaria, as well as bone samples from seven Peruvian mummies and a Bronze Age hair sample from Denmark. Prior to capture, shotgun sequencing of these libraries yielded an average of 1.2% of reads mapping to the human genome (including duplicates). After capture, this fraction increased dramatically, with up to 59% of reads mapped to human and folds enrichment ranging from 5X to 139X. Furthermore, we maintained coverage of the majority of fragments present in the pre-capture library. Intersection with the 1000 Genomes Project reference panel yielded an average of 50,723 SNPs (range 3,062-147,243) for the post-capture libraries sequenced with 1 million reads, compared with 13,280 SNPs (range 217-73,266) for the pre-capture libraries, increasing resolution in population genetic analyses. We will also present the results of performing WISC on other aDNA libraries from both archaic human and non-human samples, including ancient domestic dog samples. Our capture approach is flexible and cost-effective, allowing researchers to access aDNA from many specimens that were previously unsuitable for sequencing. Furthermore, this method has applications in other contexts, such as the enrichment of target human DNA in forensic samples.

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