Comparison of target enrichment strategies for ancient pathogen DNA

Abstract

29 In ancient DNA research, the degraded nature of the samples generally results in poor yields 30 of highly fragmented DNA, and targeted DNA enrichment is thus required to maximize 31 research outcomes. The three commonly used methods – (1) array-based hybridization capture 32 and in-solution capture using either (2) RNA or (3) DNA baits – have different characteristics 33 that may influence the capture efficiency, specificity, and reproducibility. Here, we compared 34 their performance in enriching pathogen DNA of Mycobacterium leprae and Treponema 35 pallidum of 11 ancient and 19 modern samples. We find that in-solution approaches are the 36 most effective method in ancient and modern samples of both pathogens, and RNA baits usually 37 perform better than DNA baits. 38 Method summary 39 We compared three targeted DNA enrichment strategies used in ancient DNA research for 40 the specific enrichment of pathogen DNA regarding their efficiency, specificity, and 41 reproducibility for ancient and modern Mycobacterium leprae and Treponema pallidum 42 samples. Array-based capture and in-solution capture with RNA and DNA baits were all tested 43 in three independent replicates. 44 Main Text 45 The field of ancient DNA (aDNA), which studies DNA retrieved from paleontological and 46 archaeological material, was revolutionized by the invention of high-throughput sequencing 47 (HTS). In combination with HTS, the development of targeted DNA enrichment protocols has 48 made a crucial contribution in advancing aDNA research during the last decade. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.09.195065; this version posted July 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 49 As DNA decays over time, aDNA is usually only present in trace amounts of highly 50 fragmented sequences (1, 2, 3). Detecting endogenous pathogen aDNA from archaeological 51 material is additionally compounded by the larger amount of background DNA from the 52 environment including soil microorganisms. Furthermore, the background of host DNA in 53 ancient remains is an additional obstacle in order to obtain ancient pathogen DNA. Shotgun 54 sequencing of libraries from aDNA extracts to sufficient genomic coverage is, therefore, cost- 55 intensive (4). …