DNA extraction

For paleofeces and sediment samples, DNA extractions were performed using a silica spin column protocol (Dabney et al., 2013) with minor modifications in dedicated aDNA cleanrooms located at LMAMR (Mexican paleofeces) and the MPI-SHH (all other paleofeces). At LMAMR, the modifications followed those of method D described in Hagan et al. (2020). DNA extractions at the MPI-SHH were similar, but omitted the initial bead-beating step, and a single silica column was used per sample instead of two. Additionally, to reduce centrifugation errors, DNA extractions performed at the MPI-SHH substituted the column apparatus from the High Pure Viral Nucleic Acid Large Volume Kit (Roche, Switzerland) in place of the custom assembled Zymo-reservoirs coupled to MinElute (Qiagen, Hilden, Germany) columns described in Dabney et al. (2013). Samples processed at the MPI-SHH were also partially treated with uracil-DNA-glycosylase (UDG) enzyme to confine DNA damage to the ends of the DNA molecules (Rohland et al., 2015).

For modern feces, DNA was extracted from Burkina Faso fecal samples using the AllPrep PowerViral DNA/RNA Qiagen kit at Centre MURAZ Research Institute in Burkina Faso. DNA was extracted from the Boston fecal material using the ZymoBIOMICS DNA Miniprep Kit (D4303) at the Joslin Diabetes Center.