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

[DESCRIPTION]
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]
SECTION: Buffer preparation
1. 
 

SECTION: Buffer preparation
2. 
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     
SECTION: Buffer preparation
3. 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   
SECTION: Buffer preparation
4. 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: 
3.725 mL   
45 mL   
25 µL   
1.25 mL 10 mg/ml  proteinase K solution in water (prepared from  )

 



SECTION: Sample preparation
5. In an ancient DNA cleanroom, remove approximately 50 mg  of sample powder from each specimen using a sterile dentist drill and transfer the powder to a 2.0 ml DNA LoBind tube. 

SECTION: Sample preparation
6. To facilitate resuspension of the bone powder during the subsequent incubation and wash steps, add 3-4   to the sample material. 

SECTION: Temperature-controlled phosphate treatment
7. Add 0.5 mL  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 .

 

 

SECTION: Temperature-controlled phosphate treatment
8. Transfer tubes to a tabletop centrifuge and spin for 2 min at maximum speed (e.g., 16,400g/13,200 rpm).

SECTION: Temperature-controlled phosphate treatment
9. Transfer supernatant to a 1.5 mL LoBind tube and store at -20 °C until the day of DNA extraction. 

 


SECTION: Temperature-controlled phosphate treatment
10. Repeat steps 7-9 once at each temperature (for a total of 2 wash steps).

 

SECTION: Temperature-controlled phosphate treatment
11. The temperature-controlled phosphate treatment is followed by a room-temperature wash step with 1 mL  Tris-Tween buffer at the end of the last temperature cycle. Completely resuspend the powder by thorough vortexing.

SECTION: Temperature-controlled phosphate treatment
12. Transfer tubes to a tabletop centrifuge and  spin for 2 min at maximum speed (e.g., 16,400g/13,200 rpm)
SECTION: Temperature-controlled phosphate treatment
13. Transfer supernatant to a 1.5 mL LoBind tube and store at -20 °C until the day of DNA extraction. 
SECTION: Final digestion of sample material
14. 
Add 1 mL of lysis buffer to the sample powder, completely resuspended the powder by vortexing, and incubate overnight (8 – 16 h) with rotation at 37 °C 


 



SECTION: Final digestion of sample material
15. Transfer tubes to a tabletop centrifuge and spin for 2 min at maximum speed (commonly at 16,400 g/13,200 rpm). 

SECTION: Final digestion of sample material
16. 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.
SECTION: DNA purification of phosphate fractions and final lysate
17. 
Thaw the sodium phosphate fractions (and lysates if necessary) at 37 °C  in a thermo block with gentle shaking.

 

 

SECTION: DNA purification of phosphate fractions and final lysate
18. 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.
 

SECTION: Library preparation, sequencing, and data processing
19. 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). 

 


SECTION: Library preparation, sequencing, and data processing
20. 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). 
 


SECTION: Sequence analysis
21. 
Trim adapters and merge overlapping paired-end reads into single-molecule sequences using leeHom. 
 

SECTION: Sequence analysis
22. 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.
 

 

SECTION: Sequence analysis
23. 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.
SECTION: Sequence analysis
24. Merge sequences with the same start- and end-coordinate into one consensus sequence using bam-rmdup (https://github.com/mpieva/biohazard-tools).

SECTION: Sequence analysis
25. 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.