Shotgun metagenomics applied to archaeological feces (paleofeces) can bring new insights into the composition and functions of human and animal gut microbiota from the past. However, paleofeces often undergo physical distortions in archaeological sediments, making their source species difficult to identify on the basis of fecal morphology or microscopic features alone. Here we present a reproducible and scalable pipeline using both host and microbial DNA to infer the host source of fecal

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... l. We apply this pipeline to newly sequenced archaeological specimens and show that we are able to distinguish morphologically similar human and canine paleofeces, as well as non-fecal sediments, from a range of archaeological contexts. ABSTRACT 45 PeerJ reviewing PDF | Shotgun metagenomics applied to archaeological feces (paleofeces) can bring new insights into the composition and functions of human and animal gut microbiota from the past. However, paleofeces often undergo physical distortions in archaeological sediments, making their source species difficult to identify on the basis of fecal morphology or microscopic features alone. Here we present a reproducible and scalable pipeline using both host and microbial DNA to infer the host source of fecal material. We apply this pipeline to newly sequenced archaeological specimens and show that we are able to distinguish morphologically similar human and canine paleofeces, as well as non-fecal sediments, from a range of archaeological contexts. 46 47 48 49 50 51 52 53 Previously, paleofeces host source has been genetically inferred on the basis of PCR-amplified 87 mitochondrial DNA sequences alone (Hofreiter et al., 2000); however, this is problematic in the case of 88 dogs, which, in addition to being pets and working animals, were also eaten by many ancient cultures 89 (Clutton-Brock and Hammond, 1994; Rosenswig, 2007; Kirch and O'Day, 2003; Podberscek, 2009) , and 90 thus trace amounts of dog DNA may be expected to be present in the feces of humans consuming dogs. 91 Additionally, dogs often scavenge on human refuse, including human excrement (Butler and Du Toit, 92 2002), and thus ancient dog feces could also contain trace amounts of human DNA, which could be 93 further inflated by PCR-based methods.