A method for the temperature-controlled extraction of DNA from ancient bones
Elena Essel, Matthias Meyer, Petra Korlevic
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Abstract
We here provide a protocol for the decontamination of ancient bones and teeth that is based on a temperature-controlled, sequential release of DNA. DNA can be extracted from all fractions generated with this method and the fraction with the highest proportion of endogenous DNA identified for further analysis. The protocol proceeds through repeated incubation of the sample powder in phosphate buffer at 37, 60 and 90 °C, followed by the complete lysis of the residual sample powder. As DNA is denatured at high temperature, subsequent DNA extraction and library preparation has to be performed using methods optimized for single-stranded DNA.
Steps
Buffer preparation
Sodium-phosphate buffer (0.5 M sodium phosphate, pH 7.0, 0.1 % Tween 20) is prepared by combining the following reagents:
49.5mL
50µL
Tris-Tween wash buffer (10 mM Tris-HCl, pH 8.0, 0.1% Tween-20) is prepared by combining the following reagents:
49.5mL
0.5mL
50µL
Lysis buffer (0.45 M EDTA, pH 8.0, 0.05% Tween-20 and 0.25 mg/ml proteinase K) is prepared by combining the following reagents:
3.725mL
45mL
25µL
1.25mL 10 mg/ml proteinase K solution in water (prepared from
Sample preparation
In an ancient DNA cleanroom, remove approximately 50mg of sample powder from each specimen using a sterile dentist drill and transfer the powder to a 2.0 ml DNA LoBind tube.
To facilitate resuspension of the bone powder during the subsequent incubation and wash steps, add 3-4
Temperature-controlled phosphate treatment
Add 0.5mL sodium phosphate buffer to the sample powder, completely resuspend the powder by thorough vortexing, and incubate the tube in a thermo block adjusted to the desired temperature 900rpm
Transfer tubes to a tabletop centrifuge and spin for 2 min at maximum speed (e.g., 16,400g/13,200 rpm).
Transfer supernatant to a 1.5 mL LoBind tube and store at -20 °C until the day of DNA extraction.
Repeat steps 7-9 once at each temperature (for a total of 2 wash steps).
The temperature-controlled phosphate treatment is followed by a room-temperature wash step with 1mL Tris-Tween buffer at the end of the last temperature cycle. Completely resuspend the powder by thorough vortexing.
Transfer tubes to a tabletop centrifuge and spin for 2 min at maximum speed (e.g., 16,400g/13,200 rpm)
Transfer supernatant to a 1.5 mL LoBind tube and store at -20 °C until the day of DNA extraction.
Final digestion of sample material
Add 1mL of lysis buffer to the sample powder, completely resuspended the powder by vortexing, and incubate overnight (8 – 16 h) with rotation at 37°C
Transfer tubes to a tabletop centrifuge and spin for 2 min at maximum speed (commonly at 16,400 g/13,200 rpm).
Transfer supernatant to a 1.5 mL LoBind tube and proceed to DNA extraction or store the tube at -20 °C until the day of DNA extraction.
DNA purification of phosphate fractions and final lysate
Thaw the sodium phosphate fractions (and lysates if necessary) at 37°C in a thermo block with gentle shaking.
For the sodium phosphate fractions, purify 100 µl of the supernatant, and for the final lysate, purify 500 µl using binding buffer ‘G’ of the DNA extraction method described in Glocke and Meyer (2017). Final volume of all DNA extracts is 50 µl.
Library preparation, sequencing, and data processing
Prepare DNA libraries using 20% of the DNA extract as input, following the protocol for library preparation, quantification and indexing by Gansauge et al. (2020).
Perform shallow shotgun sequencing on Illumina’s MiSeq or HiSeq2500 platforms (or other Illumina platforms) using a paired-end double-index configuration (2x 76 + 2x 7 cycles).
Sequence analysis
Trim adapters and merge overlapping paired-end reads into single-molecule sequences using leeHom.
Use the Burrows-Wheeler Aligner (BWA, https://github.com/mpieva/network-aware-bwa) to align merged sequences to a suitable reference genome (e.g. turTru1.75, bosTauUMD3.1, loxAfr4) using ancient parameters (“-n 0.01 –o 2 –l 16500”) allowing more mismatches and indels.
Restrict further analyses to sequences of length 35 bp and above to avoid spurious alignments of short sequences with random similarity to the reference genome.
Merge sequences with the same start- and end-coordinate into one consensus sequence using bam-rmdup (https://github.com/mpieva/biohazard-tools).
Generate summary statistics using samtools and choose the library with the highest proportion of endogenous DNA for further sequencing. Prepare additional libraries from remaining DNA extract if necessary.
