Raghavan et al.
July 21, 2015
Old Mission Point (ClDq-1) is located on the banks of the Restigouche River, near the town of Atholville in northern New Brunswick, Canada (Fig. S1). The site represents the prehistoric village of Tjigog, the place of summer aggregation for the northern Mi’gmaq (80-83). Rediscovered in 1968 by Martijn’s (84) archaeological surveys of Gloucester and Restigouche counties, the site was only excavated in 1972 and 1973 by Turnbull (85, 86) after construction workers unearthed human remains in a nearby gravel pit. The discovery of the burials left the skeletal assemblage badly commingled and fragmented, however, Turnbull’s (85) report features several photographs of in situ graves representing both primary and secondary internments, specifically in the form of bundle burials (see 87 and 88 for bundle burial descriptions). Artifacts recovered from the burials include a toggling harpoon head, worked bone, copper tube and shell beads, an axe head, rare pieces of cordage and braided plant-fibre textile, as well as remnants of beaver fur and birch bark (86, 88). Turnbull’s excavations also uncovered possible domestic architecture near the burial area in the form of post moulds, as well as over a thousand ceramic sherds featuring punctate, dentate-stamped, and pseudo-scallop shell designs (85). A single charcoal sample taken from a hearth feature associated with the ceramic finds gave an uncalibrated radiocarbon date of 2030±130 BP (RL-343) (89). With permission from the Listuguj Mi’gmaq community, the human remains recovered from the site underwent bioarchaeological assessment beginning in 2011 at Memorial University, where it was determined that at least 5 adults and 9 juvenile individuals (MNI=14) were included within the skeletal assemblage. Samples from a loose tooth (right mandibular first premolar (RPM1) - MARC1492) associated with a middle adult female individual (Skeleton #4) within the Old Mission Point assemblage were taken for ancient DNA analysis. The mandible of this individual in question was well-preserved unlike many of the skeletal elements found elsewhere in the assemblage, however, the in situ right first, second, and third molars (RM1, RM2, RM3) all feature a great deal of occlusal dental wear. The only remaining left molar (LM3) does not feature occlusal wear to the same extent. It is surmised that this female individual preferentially chewed on the right side of the mandible, the reason for which may be explained by the presence of a moderately healed periapical abscess located in the area of the left mandibular first and second molars (LM1, LM2) resulting in antemortem tooth loss. Uncalibrated radiocarbon AMS dates obtained on ultrafiltered bone collagen from the right femorii of 4 of the adult skeletons, (UCIAMS-125912, UCIAMS-107245, UCIAMS-107246: Skeleton #4, UCIAMS-107247) as well as the lower extremities of 4 of the juvenile skeletons (UCIAMS-125908, UCIAMS-125909, UCIAMS-125910, UCIAMS-125911) ranged from 2405-415 BP. This time range overlaps with the previous uncalibrated charcoal radiocarbon date (89), and along with the ceramic finds, suggests that Mi’gmaq individuals were living and being buried in the area as long ago as the early Middle Woodland period (ca. 2150-1650 BP) and up and until the Late Woodland (ca. 650-400 BP) or Early Historic (ca. 400-250 BP) periods (90). These findings lend support to the idea that Old Mission Point represents the oldest known long-term use Mi’gmaq cemetery in the Canadian Maritimes region to-date (88). The sampled individual was dated to ~400 BP after correcting for marine reservoir effect. (Table S2).
DNA extraction: All laboratory procedures including pre-treatment, extraction, library construction and PCR set-ups were carried out in ancient DNA facilities at the Centre for GeoGenetics (Copenhagen). Fine drill heads were used with a Dremel drill operated on low-speed setting to obtain the powdered sample. The tooth was drilled by cutting off the end of the roots and drilling into the pulp chamber with special dental drill heads, thereby collecting ~50 mg of powder. The collected powder were digested overnight at 55°C in 1ml of a buffer consisting of 1 M urea, 0.5 M EDTA and 0.3 mg/ml Proteinase K (modified from 91). Following digestion, the supernatant was concentrated using a 30 kDa centrifugal filter unit down to 100-200 μl and purified through a Qiagen MinElute spin column (using Qiagen PN binding buffer) following manufacturer’s instructions. In the elution step, the column was incubated in 45 μl of Qiagen EB buffer at 37°C for 30 minutes, spun down, and re-incubated in 30 μl of EB buffer at 37°C for 15 minutes.
Library preparation and sequencing: Two blunt-end Illumina libraries were prepared using NEBNext DNA Sample Prep Master Mix Set 2 (New England Biolabs, E6070), as described in (39), with the following differences in the protocols. Ligation was performed for 15 minutes at 20°C using Illumina-specific adapters specified in (74) and the fill-in reaction was performed for 20 minutes at 37º C. The libraries were amplified as follows: 25 μl DNA library, 1X High Fidelity PCR buffer, 2 mM MgSO4, 200 μM dNTPs each (Invitrogen, Carlsbad, CA), 200 nM Illumina Multiplexing PCR primer inPE1.0, 4 nM Illumina Multiplexing PCR primer inPE2.0, 200 nM Illumina Index PCR primer, 1 U of Platinum Taq DNA Polymerase (High Fidelity) (Invitrogen, Carlsbad, CA) and water to 50 μl. Cycling conditions were: initial denaturing at 94°C for 4 minutes, 12 cycles of: 94°C for 30 seconds, 60°C for 30 seconds, 68°C for 40 seconds, and a final extension at 72°C for 7 minutes. PCR products were purified through Qiagen MinElute spin columns and eluted in 10 μl of Qiagen Buffer EB, following a 10-minute incubation at 37°C. A second round of PCR (two parallel reactions for each library) was set up as follows: 5 μl of purified product from first PCR round, 1X High Fidelity PCR buffer, 2 mM MgSO4, 200 μM dNTPs each, 500 nM Illumina Multiplexing PCR primer 1.0, 10 nM Illumina Multiplexing PCR primer 2.0, 500 nM Illumina Index PCR primer, U of Platinum Taq DNA Polymerase (High Fidelity), and water to 50 μl. Cycling conditions included an initial denaturing at 94°C for 4 minutes, 10 cycles of: 94°C for 30 seconds, 60°C for 30 seconds, 68°C for 40 seconds, and a final extension at 72°C for minutes. Both PCR products originating from one library were purified through one Qiagen MinElute spin column and eluted in 20 μl of Qiagen Buffer EB, following a 10-minute incubation at 37°C. The amplified libraries were pooled in equimolar quantities, run on Agilent Bioanalyzer 2100 and thereafter sequenced on HiSeq 2000 (100 cycles, single-read) at the Danish National High-throughput DNA Sequencing Centre.