Computational protocol: Burying Dogs in Ancient Cis-Baikal, Siberia: Temporal Trends and Relationships with Human Diet and Subsistence Practices

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Protocol publication

[…] Bone preparation, DNA isolation and polymerase chain reaction (PCR) set-up were all performed in a dedicated, spatially isolated ancient DNA laboratory using all standard ancient DNA precautions. Negative controls were included with each extraction and PCR. DNA extraction was either with phenol-chloroform as described in or with the silica column based DNeasy kit (Qiagen). Fragments of the mitochondrial DNA were amplified in 25 µl reactions including 3 µl of extract. Reactions contained 1× buffer, 2.5 mM MgCl2, 0.2 mM dNTPs, 1 µM of each primer and 2.5 U AmpliTaq Gold (Applied Biosystems). Primers which target the ca. 425 base pairs at the 5′ end of the mitochondrial control region in three overlapping fragments were used from (size uncertainty due to presence of indels). The PCR conditions were an initial 5 min denaturation step at 95°C followed by 20 cycles of 95°C for 30 s, touchdown 0.5°C/step from 60 to 50 for 1 min, and extension at 72°C for 1 min followed by 40 cycles of 95°C for 30s, 48°C for 1 min and 72°C for 1 min with a final elongation step of 7 min at 72°C, following . Amplification was attempted from each extract at least twice for each fragment. All PCRs included negative PCR controls and the extraction negative controls. All reactions were checked on agarose gels stained with CyberSafe (Invitrogen), and all bands were directly (Sanger) sequenced in both directions with the same primers as were used in the PCR.A BLAST search ( was made with each clean sequence to determine taxa of origin, to ensure it was canid and not a common reagent contaminant such as human or cow . Sequences of canid origin were checked against replicates of the same fragment and overlapping fragments, and concatenated in Geneious . Degredation via deamination of the DNA in ancient material can yield apparent mutations. These should occur randomly, so they can be identified and resolved through replication. In the two cases where a mismatch was identified between replicates, the base pair was resolved through generation of sequences from at least one additional independent amplification. Sequences were aligned with a representative subset of previously published dog and wolf sequences , , , , in MUSCLE and a phylogenetic analysis was performed using a maximum likelihood algorithm and an HKY85 model of sequence evolution (as in ), four rate categories, and an estimated gamma parameter and transition/transversion ratio in the program PHYML . Coyote (Canis latrans) haplotypes were used to root the phylogeny . The minimum spanning network of the dog clade I haplotypes, as identified in the phylogeny, was constructed using TCS v. 1.21 . […]

Pipeline specifications

Software tools Geneious, MUSCLE, PhyML
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Canis lupus familiaris, Homo sapiens, Hemisus marmoratus, Pusa sibirica
Chemicals Carbon, Nitrogen