Feb 10, 2026
Blunt-End-Single-Tube (BEST): Double-Stranded Ancient DNA Library Protocol
- Michael V. Westbury1,
- Alba Rey-Iglesia1,
- Vanssy Li1,
- Deon de Jager1,
- Eline D. Lorenzen1
- 1University of Copenhagen
- Lorenzen Lab
Protocol Citation: Michael V. Westbury, Alba Rey-Iglesia, Vanssy Li, Deon de Jager, Eline D. Lorenzen 2026. Blunt-End-Single-Tube (BEST): Double-Stranded Ancient DNA Library Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l21yq4g1y/v1
Manuscript citation:
Carøe C, Gopalakrishnan S, Vinner L, et al. Single-tube library preparation for degraded DNA. Methods Ecol Evol. 2018; 9: 410–419. https://doi.org/10.1111/2041-210X.12871
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: October 02, 2025
Last Modified: February 10, 2026
Protocol Integer ID: 228843
Keywords: ancient DNA, double-stranded library, BEST library, dsDNA library, stranded ancient dna library protocol this prococol, stranded ancient dna library protocol, tube library preparation for degraded dna, ancient dna extraction, library build for ancient dna extraction, user ii treatment of the dna extract, dna extract, tube library preparation, degraded dna, adapter ligation, tube, protocol, stranded library, procotol, expected outcome of this procotol
Abstract
This prococol describes the process of Blunt-End-Single-Tube (BEST) library build for ancient DNA extractions. It includes USER II treatment of the DNA extract, end-repair, and adapter ligation. The expected outcome of this procotol is double-stranded libraries, which will need to be indexed before sequencing.
This protocol is originally written by Michael V. Westbury, edited by Alba Rey-Iglesia, and subsequently converted to a protocols.io version by Vanssy Li and Deon de Jager.
Figure caption from original publication: Overview of the steps in the basic library method used for all libraries in this paper. (1) Double-stranded DNA fragments are used as input. Here, a fragment containing 3′ and 5′ overhangs is presented with a uracil (U) base contained in the 5′ overhang. (2) Through end-repair, 3′ overhangs are digested by the T4 DNA Polymerase and 5′ overhangs are filled in. If the 5′ overhangs contains a uracil base, an adenosine nucleotide will be inserted on the opposite strand. Further, 3′ phosphates are removed and 5′ OH groups phosphorylated by the T4 Polynucleotide Kinase (dots). (3) Double-stranded adapters are ligated to the fragment by T4 DNA Ligase. These do not contain 5′ phosphates and therefore only one strand is ligated to the target DNA. (4) The ligated adapter is filled in and the small guiding oligo is displaced using Bst DNA Polymerase.
Carøe et al. (2018) Methods Ecol Evol, Volume: 9, Issue: 2, Pages: 410-419, First published: 17 August
2017, DOI: (10.1111/2041-210X.12871).
Always cite the original paper (Carøe et al. 2018) when using this protocol, in addition to citing this protocols.io protocol:
- Carøe C, Gopalakrishnan S, Vinner L, et al. Single-tube library preparation for degraded DNA. Methods Ecol Evol. 2018; 9: 410–419. https://doi.org/10.1111/2041-210X.12871
Materials
Reaction enhancer (for End-Repair reaction)
- PEG 4000 (Poly(ethylene glycol))Bio Basic Inc.Catalog #PB0431.SIZE.500g or 50% PEG 4000 （Mix 1g PEG4000 and 1.1 mL H20)
- BSA 20ng/mLNew England BiolabsCatalog #B9000S
- NaCl (5 M), RNase-freeThermo Fisher ScientificCatalog #10609823
- H2O
Enzymes for USER treatment
- Thermolabile USER II Enzyme - 250 unitsNew England BiolabsCatalog #M5508L
Reagents for end-repair and adapter ligase
- T4 DNA Ligase Reaction BufferNew England BiolabsCatalog #B0202S
- T4 DNA Ligase - 100,000 unitsNew England BiolabsCatalog #M0202L
- T4 DNA polymerase - 750 unitsNew England BiolabsCatalog #M0203L
- T4 PNKNew England BiolabsCatalog # M0201L
- Isothermal Amplification Buffer - 6.0 mlNew England BiolabsCatalog #B0537S
- Bst 2.0 WarmStart DNA Polymerase - 8,000 unitsNew England BiolabsCatalog #M0538L
- dNTP Set 100 mM SolutionsThermo Fisher ScientificCatalog #R0181
Buffers
- Modified Qiagen PB buffer (aka "modified binding buffer) from Allentoft et al. 2015 (DOI: 10.1038/nature14507) - see our ancient DNA extraction protocol for the recipe.
- PE buffer QiagenCatalog #19065
- Buffer EBQiagenCatalog #19086
Protocol materials
PE buffer QiagenCatalog #19065
Buffer EBQiagenCatalog #19086
T4 DNA Ligase - 100,000 unitsNew England BiolabsCatalog #M0202L
T4 DNA polymerase - 750 unitsNew England BiolabsCatalog #M0203L
PEG 4000 (Poly(ethylene glycol))Bio Basic Inc.Catalog #PB0431.SIZE.500g
BSA 20ng/mLNew England BiolabsCatalog #B9000S
dNTP Set 100 mM SolutionsThermo Fisher ScientificCatalog #R0181
NaCl (5 M), RNase-freeThermo Fisher ScientificCatalog #10609823
Thermolabile USER II Enzyme - 250 unitsNew England BiolabsCatalog #M5508L
T4 PNKNew England BiolabsCatalog # M0201L
T4 DNA Ligase Reaction BufferNew England BiolabsCatalog #B0202S
Isothermal Amplification Buffer - 6.0 mlNew England BiolabsCatalog #B0537S
Bst 2.0 WarmStart DNA Polymerase - 8,000 unitsNew England BiolabsCatalog #M0538L
50% w/v PEG 4000Molecular DimensionsCatalog #MD2-250-11
10% Tween-20 SolutionTeknovaCatalog #T0710
BSA 20 mg/mlNEBCatalog #B9000S
Troubleshooting
Before start
- This protocol should be done in an ancient DNA clean lab facility.
- Clean all surfaces with 5% bleach solution followed by 70% ethanol before and after use. Clean all equipment with 70% ethanol before and after use.
- UV-treat buffers PE, PB, and elution buffer for 10 min in a UV cross-linker before use.
- DO NOT UV enzymes or Eppendorf/PCR tubes.
- Make sure the Adapter Hybridization has been done (this only needs to be done once whenever adapters are ordered).
- UDG/USER-treat your ancient DNA extractions before starting this protocol, if this is desired. See the protocol here: USER II treatment of ancient DNA extracts.
General comments:
- When referring to "adapters" in the protocol below, we are referring to the double stranded adapter hybrid oligos (P5+P7) prepared in the adapter hybridization protocol linked above.
- Remember to include negative controls! A library build negative control, and your DNA extraction negative control.
- Keep enzymes and Master Mix in cooling block.
- Never vortex enzymes vigorously.
- Do not vortex reactions (apart from when mixing adapter and sample). Instead of vortexing, either mix by pipetting the samples 10-15 time or flick the tube with your finger 5-10 times. When handling small volumes, flicking is easier. Remember to spin down briefly prior to incubation.
- Remember to keep the master mix in a cooling block in all the time.
Buffer and adapter preparation
Reagents List:
Reaction enhancer (for end-repair reaction)
- PEG 4000 (Poly(ethylene glycol))Bio Basic Inc.Catalog #PB0431.SIZE.500g or 50% w/v PEG 4000Molecular DimensionsCatalog #MD2-250-11
- BSA 20 mg/mlNEBCatalog #B9000S
- NaCl (5 M), RNase-freeThermo Fisher ScientificCatalog #10609823
- H2O
Reagents for end-repair and adapter ligation master mixes (only those not already listed above)
- T4 DNA Ligase Reaction BufferNew England BiolabsCatalog #B0202S
- T4 DNA Ligase - 100,000 unitsNew England BiolabsCatalog #M0202L
- T4 DNA polymerase - 750 unitsNew England BiolabsCatalog #M0203L
- T4 PNKNew England BiolabsCatalog # M0201L
- Isothermal Amplification Buffer - 6.0 mlNew England BiolabsCatalog #B0537S
- Bst 2.0 WarmStart DNA Polymerase - 8,000 unitsNew England BiolabsCatalog #M0538L
- dNTP Set 100 mM SolutionsThermo Fisher ScientificCatalog #R0181
Buffers
- Modified Qiagen PB buffer (aka "modified binding buffer") from Allentoft et al. 2015 - see our ancient DNA extraction protocol for the recipe.
- PE buffer QiagenCatalog #19065
- Buffer EBQiagenCatalog #19086 or EBT buffer: 5 mL Buffer EB + 2.5 µL Tween-20 (10% Tween-20 SolutionTeknovaCatalog #T0710 )
Adapters:
- The amount of adapter (P5+P7 hybrid oligos) used is adjusted depending on the absolute amount of input DNA used in Step 5.
- Thus, ensure you have enough volume of the specific adapter concentration(s) you need before starting (1 µL needed per sample).
- Dilute the adapters as necessary from the 200 µM stock prepared in the adapter hybridization protocol if you do not have enough.
| DNA input amount (ng) | Corresponding adapter concentration (µM) | |
| 100-400* | 50 | |
| 50-100 | 20 | |
| 30-50 | 10 | |
| 20-30 | 5 | |
| 10-20 | 3 | |
| 5-10 | 2 | |
| <5** | 1 |
Note
*100-400 ng: Rather than using 1 μL of 50 μM adapter, it is recommended to dilute the DNA extraction to 50-100 ng and use the 20 μM adapter. The BEST protocol is optimal for DNA input between 0.1 and 200 ng.
**<5 ng: Too little DNA.
- You will use a maximum of 16 μL of your DNA extraction (Step 5), so: 16 μL x DNA concentration in ng/μL = ng of DNA used.
- If your DNA extract is too concentrated (i.e. you have >400 ng in 16 uL, then dilute your DNA with EB buffer (also see * above).
Reaction enhancer (for end-repair reaction):
- Prepare in advance. The mixture can be frozen and mixed with the master mix buffer in the end-repair reaction (Step 4).
| Reagent | Stock Conc. | Volume | Final Conc. | |
| 50% PEG 4000* | 50% | 500 uL | 25% | |
| BSA | 20 mg/mL | 100 μL | 2 mg/mL | |
| NaCl | 5 M | 80 μL | 400 mM | |
| H2O | / | up tp 1 mL | / | |
| Final | / | 1 mL | / |
*Or 0.25 g of powdered PEG4000.
End-repair
Prepare the end-repair master mix.
| Reagent | Stock Conc. | Final Conc.* | Volume | N samples | |
| T4 DNA polymerase | 3 U/μL | 0.03 U/μL | 0.2 μL | 0.2 μL*(N+1) | |
| T4 PNK | 10 U/μL | 0.25 U/μL | 0.5 μL | 0.5 μL*(N+1) | |
| dNTP | 25 mM | 0.25 mM | 0.2 μL | 0.2 μL*(N+1) | |
| T4 DNA ligase buffer | 10x | 1x | 2 μL | 2 μL*(N+1) | |
| Reaction enhancer | / | / | 1.1 μL | 1.1 μL*(N+1) | |
| Final | / | / | 4 μL | 4 μL*(N+1) |
*Note: This final concentration refers to the concentration in the total reaction in Step 5 (final volume of 20 µL), and not the final concentration in the end-repair master mix itself.
In PCR tubes (individual or a strip), add 4 μL of the end-repair master mix to 16 μL DNA (may or may not be USER-treated) for each sample, for a total reaction volume of 20 µL.
In a thermocycler, incubate the reaction for 00:30:00 min at 20 °C followed by 00:30:00 min at 65 °C and then cool to 4 °C
Note
It is important that the reaction is cooled to 4°C for complete deactivation.
During this incubation (~1 h), prepare the ligation master mix (Step 8, below), and put it in a cold rack or on ice before using it.
Once the end-repair incubation is complete, put the PCR tubes in a cold rack (or ice) and add 1 μL adapter to each reaction, ensuring to use the concentration that corresponds to the amount of input DNA in the reaction (see Step 2). The reaction volume is now 21 µL.
- Mix thoroughly by pipetting or by flicking and spinning down in a microcentrifuge several times.
Adapter ligation
Prepare the ligation master mix
| Reagent | Stock Conc. | Final Conc.* | Volume | N samples | |
| T4 DNA ligase buffer | 10x | 0.2x | 0.5 μL | 0.5 μL*(N+1) | |
| 50% PEG 4000 | 50% | 6% | 3 μL | 3 μL*(N+1) | |
| T4 DNA ligase | 400 U/μL | 8 U/μL | 0.5 μL | 0.5 μL*(N+1) | |
| Final | / | / | 4 μL | 4 μL*(N+1) |
*Note: This final concentration refers to the concentration in the total reaction in Step 9 (final volume of 25 µL), and not the final concentration in the ligation master mix itself.
Add 4 μL of the ligation master mix to each sample to make a 25 μL reaction.
Incubate for 00:30:00 min at 20 °C followed by 00:10:00 min at 65 °C and then cool to 4 °C .
During this incubation (~40 min), prepare the adapter fill-in master mix (Step 11, below), and put the master mix in a cold rack or on ice until it is needed.
Adapter fill-in
Prepare the adapter fill-in master mix.
| Reagent | Stock Conc. | Final Conc.* | Volume | N samples | |
| Isothermal amp. buffer | 10x | 0.33x | 1 μL | 1 μL*(N+1) | |
| dNTP | 25 mM | 0.33 mM | 0.4 μL | 0.4 μL*(N+1) | |
| Bst 2.0 Warmstart pol | 8 U/μL | 0.21 U/μL | 0.8 μL | 0.8 μL*(N+1) | |
| H2O | / | / | 2.8 μL | 2.8 μL*(N+1) | |
| Final | 5 μL | 5 μL*(N+1) |
*Note: This final concentration refers to the concentration in the total reaction in Step 12 (final volume of 30 µL), and not the final concentration in the adapter fill-in master mix itself.
Add 5 μL of the adapter fill-in master mix to each sample to make a 30 μL reaction.
Incubate for 00:15:00 min at 65 °C followed by 00:15:00 min at 80 °C and then cool to 4 °C .
During this incubation (~30 min), prepare the tubes for the DNA clean-up. Label Monarch columns (temporary) and one set of final 1.5 mL LoBind Eppendorf tubes. Label appropriately with sample code, date, your name etc. Also prepare a 50 mL Falcon tube and label it as "Qiagen waste" and with your name and the date.
Proceed to clean-up (Step 14) immediately after incubation.
Clean up using monarch spin columns
Note
REMINDER: UV-treat buffers for 10 min in a UV cross-linker before use, if not already done.
After the adapter fill-in incubation is complete, add 450 μL modified PB (binding) buffer to the Monarch spin columns (one for each sample).
Add the entire adapter fill-in reaction (30 μL) to the modified PB (binding) buffer in the spin column and mix by pipetting.
Centrifuge at 8.000 rpm for 00:01:00 min.
- Prepare a square piece of paper towel/Kim Wipe by folding it in half twice (so there are four layers).
- Discard the flow through into the 50 mL Falcon tube labelled as "Qiagen waste".
- Dry the collection tube by tapping it upside down on the folded piece of paper towel before putting the column back. Ensure that you tap on a different piece of the paper towel for each sample, to avoid cross-contamination.
Add 650 μL PE buffer to each spin column and centrifuge at 8.000 rpm for 00:01:00 min.
Discard the flow through and dry the collection tube as before.
Centrifuge at maximum speed for 00:01:00 min to dry the spin column of any excess PE buffer.
DNA (library) elution: Place spin column in a fresh 1.5 mL LoBind Eppendorf tube and add 16 μL EBT buffer (or EB buffer) to the center of the spin column membrane.
Note
If LoBind tubes are not available, it is essential to use EBT or TET buffer (see the SCR protocol) in this step, as this helps to prevent the binding of DNA to the plastic of the tube.
It is obviously crucial to retain as much DNA as possible when working with aDNA libraries due to the low concentration and complexity of the libraries and the fact the source of the DNA is usually finite, so every step should limit DNA loss as much as possible.
When using LoBind tubes, standard Buffer EB can be used (without Tween-20), as the LoBind tubes are designed to reduce the binding of DNA. Although note that a recent study (Gilardet et al. 2025) showed that using TET (which includes Tween-20) for elution of aDNA extractions increases complexity of the final library - granted, that was for extractions (and used TET and not EBT), but it should be effective for libraries too.
Incubate for 00:05:00 and then centrifuge at maximum speed for 00:01:00 min to collect the eluted DNA in the Eppendorf tube.
Repeat steps 19 and 20 to give a final library volume of 32 μL.
Store libraries at -20°C.
Protocol references
Carøe C, Gopalakrishnan S, Vinner L, et al. Single-tube library preparation for degraded DNA. Methods Ecol Evol. 2018; 9: 410–419. https://doi.org/10.1111/2041-210X.12871