Jul 25, 2025
DNA Extraction and mtDNA genome sequencing protocol from museum voucher specimens
- M. Alejandra Camacho1,
- SANTIAGO F. BURNEO1,
- Balázs orváth2,
- Dániel adar2,
- Gábor óth3,
- JÉRÔME RIENNE4
- 1Museo de Zoología, Facultad de Ciencias Exactas y Naturales, Pontificia Universidad Católica del Ecuador, Quito, Ecuador;
- 2WHO Collaborating Centre for Arbovirus and Haemorrhagic Fever Reference and Research, Bernhard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany;
- 3Bernhard Nocht Institute for Tropical Medicine;
- 4Centre de Recherche sur la Biodiversité et l'Environnement (CRBE UMR5300), Université de Toulouse, CNRS, IRD, Toulouse INP, Université Toulouse 3 – Paul Sabatier (UT3), Toulouse, France
Protocol Citation: M. Alejandra Camacho, SANTIAGO F. BURNEO, Balázs orváth, Dániel adar, Gábor óth, JÉRÔME RIENNE 2025. DNA Extraction and mtDNA genome sequencing protocol from museum voucher specimens. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8q3r7l2w/v1
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: In development
We are still developing and optimizing this protocol
Created: June 20, 2025
Last Modified: July 25, 2025
Protocol Integer ID: 220628
Keywords: voucher, museum specimens, mitochondrion, NGS, sequencing, sequencing mitochondrial genome, mitochondrial dna, mitochondrial dna versus morphology, mitochondrial genome, molecular data from authenticated voucher specimen, museum voucher specimen, taxonomic assessment, mtdna, archived specimen, taxonomic resolution, retrospective species identification, reconstructing mammalian phylogeny, invaluable resource for biodiversity research, voucher specimen, genetic analyses from ancient dna, verifiable records of species identity, based taxonomic, molecular phylogenetics, phylogenetic study, authenticated voucher specimen, dna extraction, mammalian phylogeny, biodiversity research, ancient dna, protocol from museum voucher, species identity, museum voucher, genetic marker, museum collection, value of museum collection, species, dna, genetic analysis, mammal sample, conservation assessment, annual review of genetics, reproducibility across study, museum, sequencing protocol
Abstract
Museum voucher specimens represent an invaluable resource for biodiversity research, serving as permanent, verifiable records of species identities, morphology, and provenance (Suarez & Tsutsui, 2004). In recent decades, these archived specimens have also become critical molecular reservoirs, particularly for DNA-based taxonomic and phylogenetic studies (Wandeler et al., 2007). Among genetic markers, mitochondrial DNA (mtDNA) is especially suited for taxonomic assessment due to its maternal inheritance, relatively rapid mutation rate, and high copy number per cell, which enhances its retrievability even from degraded or historical samples (Avise, 2000; Paabo et al., 2004).
In mammals, sequencing mitochondrial genomes or key regions (e.g., cytochrome b, COI) from voucher specimens enables retrospective species identification, clarifies cryptic diversity, and facilitates phylogeographic and conservation assessments (Tobe et al., 2010; Lorenzen et al., 2011). Integrating molecular data from authenticated voucher specimens strengthens taxonomic resolution and provides reproducibility across studies, underscoring their dual importance in both classical and modern systematics.
Our protocol offers multiple extraction methods for variously stored samples (formallin or ethanol fixed). It makes it widely usable but the method was mainly tested on mammal samples.
References
- Avise, J. C. (2000). Phylogeography: The History and Formation of Species. Harvard University Press.
- Lorenzen, E. D., et al. (2011). Species-specific responses of Late Quaternary megafauna to climate and humans. Nature, 479(7373), 359–364.
- Paabo, S., et al. (2004). Genetic analyses from ancient DNA. Annual Review of Genetics, 38, 645–679.
- Suarez, A. V., & Tsutsui, N. D. (2004). The value of museum collections for research and society. BioScience, 54(1), 66–74.
- Tobe, S. S., Kitchener, A. C., & Linacre, A. M. T. (2010). Reconstructing mammalian phylogenies: mitochondrial DNA versus morphology. Molecular Phylogenetics and Evolution, 55(2), 727–733.
- Wandeler, P., Hoeck, P. E., & Keller, L. F. (2007). Back to the future: museum specimens in population genetics. Trends in Ecology & Evolution, 22(12), 634–642.
Materials
| A | B | C | |
| Reagents | |||
| Component | Manufacturer | Catalog number | |
| DNeasy Blood and Tissue Kit or | Qiagen | 69504 | |
| MinElute Reaction Cleanup Kits | Qiagen | 28204 | |
| Absolute Ethanol (Molecular Biology Grade) | any | - | |
| Phenol (Molecular Biology Grade) | - | ||
| Isopropanol(Molecular Biology Grade) | any | - | |
| Sodium acetate (Molecular Biology Grade) | any | - | |
| SDS | any | - | |
| NaOH | any | - | |
| QIAseq FX DNA Library UDI Kit (24) | Qiagen | 180477 | |
| Qubit™ 1X dsDNA High Sensitivity (HS) | Invitrogen | Q33230 | |
| Nuclease free water | any | - | |
| Agencourt® AMPure® XP | Beckman Coulter | A63880 | |
| apeStation DNA ScreenTape & Reagents | Agilent | - | |
| Flow cell | Illumina | - | |
| Cartridge | Illumina | - | |
| A | B | C | |
| Consumables | |||
| Component | Manufacturer | Catalog number | |
| Qubit™ Assay Tubes | Invitrogen | Q32856 | |
| 1.5 ml tubes | any | - | |
| Pipette tips 0.1-1000 μl | suitable | - | |
| PCR tubes 0.2 ml | any | - | |
| A | B | C | |
| Equipments | |||
| Component | Manufacturer | Catalog number | |
| Pipettes 0.1-1000 μl | any | - | |
| Centrifuge | any | - | |
| Magnetic stand or rack | any | - | |
| Qubit™ 4 Fluorometer | Invitrogen | Q33238 | |
| End-point PCR machines | any | - | |
| Tapestation System (4150 or 4200) | Agilent | - | |
| Illumina sequencer | Illumina | - | |
Troubleshooting
Safety warnings
Use all reagents based on the manufacturer's information, and check the risks and countermeasures on the safety data sheet!
Before start
If you are using the Phenol-Chloroform extraction, prepare the solutions in advance. I case of kits the handbook contains the necessary preparation before operation.
DNA Extraction from Formalin-fixed Tissue
2d 6h 54m 15s
For this version of extraction the DNeasy Blood and Tissue Kit (Qiagen) is used.
Manually chop the tissue and soak in water at room temperature for 06:00:00 hours than remove the water.
6h
Add 300 µL ATL buffer (pre-warmed to 98 °C ) to the tissue and incubate at 98 °C for 00:15:00 minutes in a heating block.
15m
Cool down the samples on ice for 00:02:00 minutes.
2m
Add 40 µL Proteinase K and incubate for 48:00:00 hours at 65 °C .
2d
If the tissue is not digested completely, Proteinase K can be added up to 3 more times in 25 µL increments.
Vortex the sample for 00:00:15 seconds and briefly spin it down.
15s
Split the sample into two tubes and proceed with both (load both on the same column).
Add 200 µL AL buffer (for both aliquot), vortex and centrifuge.
Add 200 µL Ethanol (96–100%), vortex thoroughly and centrifuge.
Load both samples onto the same column, spin at 8000 rpm for 00:01:00 min.
1m
Discard collection tube, place column in a new collection tube.
Add 500 µL AW1 buffer, spin at 8000 rpm for 00:01:00 min.
1m
Discard collection tube, place column in a new collection tube.
Add 500 µL AW2 buffer, spin at 14,000 rpm for 00:03:00 min.
3m
Place column in a new collection tube, spin empty at 14,000 rpm for 00:01:00 min.
1m
Place column in a new 1.5 mL tube and add 140 µL AE buffer to the column.
Incubate for 00:30:00 min at room temperature, spin at 8000 rpm for 00:01:00 min.
Note
For more information you can found the kit handbook here:
HB-2061-004_HB_DNY_Blood_Tissue_0623_WW.pdf 31m
If you used this method for DNA extraction, go to Step 4 and proceed with quality check.
DNA Extraction using Phenol-Chloroform
1h 45m
For this version of extraction the classical Phenol-Chloroform method is used.
Add 500 µL Alkaline lysis buffer (0.1 M NaOH, 1% SDS) to 1.5 mL screw-cap Eppendorf tubes with O-ring.
Add tissue piece and incubate at 100 °C for 00:40:00 minutes.
40m
Cool down for 00:05:00 at room temperature.
5m
Add 500 µL Phenol/Chloroform/Isoamyl alcohol (25:24:1), mix gently for 00:05:00 min.
5m
Centrifuge at 10,000 rpm for 00:05:00 min.
5m
Transfer the upper aqueous phase to a fresh tube and add 500 µL Chloroform.
Mix gently for 00:05:00 min and centrifuge again at 10,000 rpm for 00:05:00 min.
10m
Carefully transfer the upper aqueous phase and precipitate the DNA by adding 0.6–1x volume Isopropanol and 0.1x volume 3 Molarity (M) Sodium acetate.
Centrifuge at 13,000 g for 00:30:00 min at room temperature. Remove the supernatant carefully.
30m
Wash the pellet with 500-1000 µL 85% Ethanol, centrifuge at 13,000 g for 00:05:00 min.
5m
Remove ethanol completely and carefully. Dry the pellet with open cap for 00:05:00 min.
5m
Resuspend the pellet in 50 µL Nuclease-free water.
If you used this method for DNA extraction, go to Step 4 and proceed with quality check.
DNA Extraction from Ethanol-fixed Tissue (MinElute Kit)
2d 0h 33m
For this version of extraction the DNeasy Blood and Tissue Kit (Qiagen) is used.
Wash tissue pieces 3 times with 400 µL nuclease free water for 00:30:00 minutes.
30m
Remove the water from the last washing step.
Incubate tissue sample with 180 µL ATL lysis buffer (Qiagen) and 20 µl Proteinase K at 55 °C for 48:00:00 hours in a heat block.
2d
Add 300 µL ERC buffer to each portion of 100 µL lysate.
Buffer mix should appear yellow (7.5 ); if not, add 10 µL 3 Molarity (M) Sodium acetate.
Transfer the sample to a MinElute spin column.
Centrifuge at 10,000 g for 00:01:00 min. Discard collection tube and reuse the column with a new sample portion (if applicable).
1m
Wash bound DNA with 750 µL PE buffer. Centrifuge at 10,000 g for 00:01:00 min.
1m
Discard flow-through and centrifuge the empty column at maximum speed. Place column into a new 1.5 mL tube.
Elute DNA with 50 µL EB buffer, incubate 1 min at room temperature and centrifuge at 10,000 g for 00:01:00 min.
Note
For more information you can found the kit handbook here:
EN-MinElute-Handbook.pdf 1m
Quality check
2m
For quality check the concentration was measured with Qubit fluorometer using the 1xds High Sensitivity kit.
Aliquot 199 µL 1x Working solution to Qubit assay tube (equal to the number of your samples). Prepare two extra tubes for the standards with 190 µL 1x Working solution.
Add 1 µL sample for each test tube and 10 µL from the Standards (Standard 1, Standard 2). The final volume is 200 µL
Mix each sample vigorously by vortexing and pulse centrifuge to collect the liquid. Incubate at room temperature for 00:02:00 min before measuring.
2m
Calibrate Qubit fluorometer with the standards based on manufacturers recommendation.
Read your sample.
Note
If it is possible, fragment length could be checked on Bioanalyzer or Tapestation system.
NGS Library preparation
1d 1h 3m 20s
For the library preparation we use the QIAseq FX DNA Library Kit (Qiagen)
Note
The handbook of FX DNA Library Kit.
HB-2015-006_HB_QIAseq_FX_DNA_Library_0624_WW.pdf Sample dilution
We need 100 ng DNA input for library preparation. Dilute the sample with Nuclease-free water to 100 ng /35 µL concentration.
Fragmentation and End preparation
Prepare the following reaction:
Component Volume
FX Buffer 10x 5 µL
FX Enzyme Mix 10 µL
Sample DNA (100 ng ) 35 µL
Final volume: 50 µL
Incubate the reaction with the following conditions:
Temperature Time
4 °C 00:01:00
32 °C 00:01:00
65 °C 00:30:00
4 °C
Note
DNA fragmentation was minimised due to the high degradation of nucleic acid (DNA ≤ 500 bp) material and low concentration (≤ 3 ng /µl).
32m
Adapter Ligation
Prepare the following reaction:
Component Volume
DNA-Ligase Buffer 5x 20 µL
DNA-Ligase 10 µL
Nuclease free H2O 15 µL
Unique Adapter 5 µL
Previous reaction 50 µL
Final volume: 100 µL
Incubate at 20 °C for 00:15:00 minutes.
15m
Clean up
Clean up the adapter ligated product with Ampure Xp Beads
Mix 80 µL beads with the previous reaction and incubate for 00:05:00 minutes at room temperature.
5m
Place on magnetic stand for 00:02:00 and carefully remove the supernatant.
2m
Wash the beads with 200 µL 80% EtOH for 00:00:30 seconds and and carefully remove the supernatant.
30s
Repeat the washing step: Wash the beads with 200 µL 80% EtOH for 00:00:30 seconds and and carefully remove the supernatant.
30s
Allow the beads to dry on the magnetic stand for approx. 5 min (till the point when the pellet lose its shine).
Add 52.5 µL elution buffer to the dried beads and resuspend well. Incubate for 00:02:00 minutes off the magnetic stand. Place back to the magnetic stand and incubate for additional 00:02:00 .
4m
Remove 50 µL of the supernatant and transfer to a new PCR tube.
Library amplification
Prepare the following reaction
Component Volume
HIFI PCR Master Mix 2x 25 µL
adapter Primer Mix 1.5 µL
Cleaned up DNA 23.5 µL
Final volume 50 µL
Reaction conditions:
Temperature Time No. of cycles
98 °C 00:02:00 1
98 °C 00:00:20 8
60 °C 00:00:30 8
72 °C 00:00:30 8
72 °C 00:01:00 1
4 °C Till further processing
4m 20s
Library Clean up
Clean up the amplified library with Ampure Xp Beads
Mix 50 µL beads with the previous reaction and incubate for 00:05:00 minutes at room temperature.
Place on magnetic stand for 00:02:00 and carefully remove the supernatant.
Wash the beads with 200 µL 80% EtOH for 00:00:30 seconds and and carefully remove the supernatant.
Repeat the washing step: Wash the beads with 200 µL 80% EtOH for 00:00:30 seconds and and carefully remove the supernatant.
Allow the beads to dry on the magnetic stand for approx. 5 min (till the point when the pellet lose its shine).
Add 52.5 µL elution buffer to the dried beads and resuspend well. Incubate for 00:02:00 minutes off the magnetic stand. Place back to the magnetic stand and incubate for additional 00:02:00 .
Remove 50 µL of the supernatant and transfer to a new PCR tube. Storage at 4 °C for a day is possible but for long term storage -20 °C is suitable.
1d
Library Quantification and checking
Measure the concentration with Qubit Fluorometer according to Step 4. If it is possible fragment length could be checked on Bioanalyzer or Tapestation system. Alternative solution is gel electrophoresis (1.5% gel), as the library concentration should be high enough.
Start sequencing
Pool the sequencing based on the applied flow cell and equipment and start the run. Mitochondrial DNA typically constitutes <0.5–1% of total reads in whole-genome Illumina shotgun libraries. The minimum sequencing depth for successful recovery of complete mitochondrial genome is around 10 million reads/sample.