TITLE: A method for the temperature-controlled extraction of DNA from ancient bones 
AUTHORS: Elena  Essel, Matthias Meyer, Petra Korlevic

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[STEPS]
?. [Buffer preparation]
All buffers are irradiated with UV-C light at a dose of 7 kJ/cm2 using a cross-linker.
?. [Buffer preparation]
Sodium-phosphate buffer (0.5 M sodium phosphate, pH 7.0, 0.1 % Tween 20) is prepared by combining the following reagents:
49.5 mL
50 µl
?. [Buffer preparation]
Tris-Tween wash buffer (10 mM Tris-HCl, pH 8.0, 0.1% Tween-20) is prepared by combining the following reagents:
49.5 mL
0.5 mL
50 µl
?. [Buffer preparation]
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:  10 mg/ml  proteinase K solution in water (prepared from )
3.725 mL
45 mL
25 µl
1.25 mL
Proteinase K is added after UV irradiation
?. [Sample preparation]
In an ancient DNA cleanroom, remove approximately   of sample powder from each specimen using a sterile dentist drill and transfer the powder to a 2.0 ml DNA LoBind tube.
50 mg
?. [Sample preparation]
To facilitate resuspension of the bone powder during the subsequent incubation and wash steps, add 3-4  to the sample material.
?. [Temperature-controlled phosphate treatment]
Add   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
0.5 mL
33
Temperature-controlled phosphate treatment steps  2 times  2 times  2 times
At least one negative control (tube without sample material) should be included in each experiment and carried through all subsequent steps).
?. [Temperature-controlled phosphate treatment]
Transfer tubes to a tabletop centrifuge and spin for 2 min at maximum speed (e.g., 16,400g/13,200 rpm).
?. [Temperature-controlled phosphate treatment]
Transfer supernatant to a 1.5 mL LoBind tube and store at -20 °C until the day of DNA extraction.
Beads facilitate the resuspension of the sample powder after centrifugation steps, but make it harder to remove supernatant.Pipette slowly and carefully.
?. [Temperature-controlled phosphate treatment]
Repeat steps 7-9 once at each temperature (for a total of 2 wash steps).
For the 90 °C incubation, make sure the liquid in the tube reaches 90 °C by the end of the 15 min incubation time. If necessary, set the thermo block to a higher temperature.
?. [Temperature-controlled phosphate treatment]
The temperature-controlled phosphate treatment is followed by a room-temperature wash step with   Tris-Tween buffer at the end of the last temperature cycle. Completely resuspend the powder by thorough vortexing.
1 mL
?. [Temperature-controlled phosphate treatment]
Transfer tubes to a tabletop centrifuge and  spin for 2 min at maximum speed (e.g., 16,400g/13,200 rpm)
?. [Temperature-controlled phosphate treatment]
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  of lysis buffer to the sample powder, completely resuspended the powder by vortexing, and incubate overnight (8 – 16 h) with rotation at
1 mL
37 °C
Wrap the tube with parafilm to prevent leaking.
?. [Final digestion of sample material]
Transfer tubes to a tabletop centrifuge and spin for 2 min at maximum speed (commonly at 16,400 g/13,200 rpm).
?. [Final digestion of sample material]
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   in a thermo block with gentle shaking.
37 °C
Make sure the liquid is fully thawed and any crystals have completely dissolved.
If desired, DNA extraction can also be performed on the Tris-Tween buffer, but DNA yields are expected to be extremely low.
?. [DNA purification of phosphate fractions and final lysate]
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). 
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?. [Library preparation, sequencing, and data processing]
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. 
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?. [Sequence analysis]
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.
?. [Sequence analysis]
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.
?. [Sequence analysis]
Merge sequences with the same start- and end-coordinate into one consensus sequence using bam-rmdup (https://github.com/mpieva/biohazard-tools).
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?. [Sequence analysis]
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.