Sep 07, 2023
Version 2
A method for the temperature-controlled extraction of DNA from ancient bones V.2
Version 1 is forked from A method for the temperature-controlled extraction of DNA from ancient bones
- Elena Essel1,
- Petra Korlevic1,2,
- Matthias Meyer1,2
- 1Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, D-04103 Leipzig, Germany;
- 2EMBL-EBI, Wellcome Genome Campus, Hinxton, Cambridgeshire, CB10 1SD, UK
- MPI EVA Ancient DNA Core Unit
Protocol Citation: Elena Essel, Petra Korlevic, Matthias Meyer 2023. A method for the temperature-controlled extraction of DNA from ancient bones . protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz3pb2gx1/v2Version created by Elena Essel
Manuscript citation:
A method for the temperature-controlled extraction of DNA from ancient bones Elena Essel, Petra Korlević, and Matthias Meyer BioTechniques 2021 71:1, 382-386 https://doi.org/10.2144/btn-2021-0025
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 07, 2023
Last Modified: April 10, 2024
Protocol Integer ID: 87493
Keywords: Ancient DNA, sequential DNA extraction, contamination removal, endogenous DNA, archaeological material, dna extraction, extraction of dna, subsequent dna extraction, sequential release of dna, stranded dna, dna, ancient bone, endogenous dna, protocol for the decontamination, incubation of the sample powder, controlled extraction, extraction, decontamination, temperature, sample powder, complete lysis of the residual sample powder, phosphate buffer, high temperature
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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.
Materials
Reagents
Sodium phosphate, 0.5M buffer soln., pH 7.0 Thermo ScientificCatalog #AAJ63791AP
Water for HPLCMerck MilliporeSigma (Sigma-Aldrich)Catalog #270733
EDTA solution pH 8.0 (0.5 M) for molecular biologyAppliChemCatalog #A4892,1000
Tris buffer pH 8.0 (1 M) for molecular biologyAppliChemCatalog #A4577,0500
Proteinase K 100 mgMerck MilliporeSigma (Sigma-Aldrich)Catalog #3115879001
TWEEN® 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #T2700-100ML
Consumables and equipment
DNA LoBind Tubes 2.0 mLEppendorfCatalog #0030108078
DNA LoBind Tubes 2.0 mLEppendorfCatalog #0030108078
Ceramic beads 2.8 mm VWR International (Avantor)Catalog #432-0292
50 ml CELLSTAR® Polypropylene Tube 30/115 MM Conical Bottom Blue screw cap sterile skirtgreiner bio-oneCatalog #210261
Parafilm M 10 cm widneoLabCatalog #3-1012
Equipment
Thermomixer
NAME
HLC
BRAND
52 82 00133
SKU
Equipment
Incubator
NAME
Memmert
BRAND
Incubator IN55
SKU
Equipment
Tube rotator
NAME
VWR
BRAND
444-0500
SKU
Equipment
UV cross-linker
NAME
Vilber
BRAND
Bio-Link BLX 254
SKU
Equipment
Vortex mixer
NAME
Scientific Industries
BRAND
SI-0236
SKU
Equipment
Centrifuge
NAME
Bench centrifuge
TYPE
Eppendorf
BRAND
5424
SKU
Protocol materials
Proteinase K 100 mgMerck MilliporeSigma (Sigma-Aldrich)Catalog #3115879001
50 ml CELLSTAR® Polypropylene Tube 30/115 MM Conical Bottom Blue screw cap sterile skirtgreiner bio-oneCatalog #210261
Tris buffer pH 8.0 (1 M) for molecular biologyAppliChemCatalog #A4577,0500
TWEEN® 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #T2700-100ML
Ceramic beads 2.8 mm VWR International (Avantor)Catalog #432-0292
Sodium phosphate, 0.5M buffer soln., pH 7.0 Thermo ScientificCatalog #AAJ63791AP
Water for HPLCMerck MilliporeSigma (Sigma-Aldrich)Catalog #270733
Parafilm M 10 cm widneoLabCatalog #3-1012
DNA LoBind Tubes 2.0 mLEppendorfCatalog #0030108078
EDTA solution pH 8.0 (0.5 M) for molecular biologyAppliChemCatalog #A4892,1000
Buffer preparation
Note
All buffers are irradiated with UV-C light at a dose of 7 kJ/cm2 using a cross-linker.
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 Sodium phosphate, 0.5M buffer soln., pH 7.0 Thermo ScientificCatalog #AAJ63791AP
50 µL TWEEN® 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #T2700-100ML
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 Water for HPLCMerck MilliporeSigma (Sigma-Aldrich)Catalog #270733
0.5 mL Tris buffer pH 8.0 (1 M) for molecular biologyAppliChemCatalog #A4577,0500
50 µL TWEEN® 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #T2700-100ML
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 Water for HPLCMerck MilliporeSigma (Sigma-Aldrich)Catalog #270733
45 mL EDTA solution pH 8.0 (0.5 M) for molecular biologyAppliChemCatalog #A4892,1000
25 µL TWEEN® 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #T2700-100ML
1.25 mL 10 mg/ml proteinase K solution in water (prepared from Proteinase K 100 mgMerck MilliporeSigma (Sigma-Aldrich)Catalog #3115879001 )
Note
Proteinase K is added after UV irradiation
Sample preparation
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.
To facilitate resuspension of the bone powder during the subsequent incubation and wash steps, add 3-4Ceramic beads 2.8 mm VWR International (Avantor)Catalog #432-0292 to the sample material.
Temperature-controlled phosphate treatment
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 900 rpm, 00:15:00.
Note
Temperature-controlled phosphate treatment steps
37 °C 2 times
60 °C 2 times
90 °C 2 times
Note
At least one negative control (tube without sample material) should be included in each experiment and carried through all subsequent steps).
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.
Note
Beads facilitate the resuspension of the sample powder after centrifugation steps, but make it harder to remove supernatant.
Pipette slowly and carefully.
Repeat steps 7-9 once at each temperature (for a total of 2 wash steps).
Note
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.
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.
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 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
Note
Wrap the tube with parafilm to prevent leaking.
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.
Note
Make sure the liquid is fully thawed and any crystals have completely dissolved.
Note
If desired, DNA extraction can also be performed on the Tris-Tween buffer, but DNA yields are expected to be extremely low.
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.
Citation
LINK
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).
Citation
LINK
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).
Citation
LINK
Sequence analysis
Trim adapters and merge overlapping paired-end reads into single-molecule sequences using leeHom.
Citation
LINK
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.
Citation
LINK
Citation
LINK
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.
Citation
LINK
Citations
Step 18
Glocke I, Meyer M. Extending the spectrum of DNA sequences retrieved from ancient bones and teeth.
https://doi.org/10.1101/gr.219675.116Step 19
Gansauge MT, Aximu-Petri A, Nagel S, Meyer M. Manual and automated preparation of single-stranded DNA libraries for the sequencing of DNA from ancient biological remains and other sources of highly degraded DNA.
https://doi.org/10.1038/s41596-020-0338-0Step 20
Kircher M, Sawyer S, Meyer M. Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform.
https://doi.org/10.1093/nar/gkr771Step 21
Renaud G, Stenzel U, Kelso J. leeHom: adaptor trimming and merging for Illumina sequencing reads.
https://doi.org/10.1093/nar/gku699Step 22
Meyer M, Kircher M, Gansauge MT, Li H, Racimo F, Mallick S, Schraiber JG, Jay F, Prüfer K, de Filippo C, Sudmant PH, Alkan C, Fu Q, Do R, Rohland N, Tandon A, Siebauer M, Green RE, Bryc K, Briggs AW, Stenzel U, Dabney J, Shendure J, Kitzman J, Hammer MF, Shunkov MV, Derevianko AP, Patterson N, Andrés AM, Eichler EE, Slatkin M, Reich D, Kelso J, Pääbo S. A high-coverage genome sequence from an archaic Denisovan individual.
https://doi.org/10.1126/science.1224344Step 22
Li H, Durbin R. Fast and accurate long-read alignment with Burrows-Wheeler transform.
https://doi.org/10.1093/bioinformatics/btp698Step 25
Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, 1000 Genome Project Data Processing Subgroup.. The Sequence Alignment/Map format and SAMtools.
https://doi.org/10.1093/bioinformatics/btp352