Nov 07, 2025

Public workspaceTargeted hybridization capture for phylogenomics

DOI
https://dx.doi.org/10.17504/protocols.io.e6nvw12jzlmk/v1
  • Sabine Nidelet1,
  • Jean-Yves Rasplus1,
  • Astrid Cruaud1
  • 1INRAE-CBGP
  • INRAE
Sabine Nidelet
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DOI: https://dx.doi.org/10.17504/protocols.io.e6nvw12jzlmk/v1
Protocol Citation: Sabine Nidelet, Jean-Yves Rasplus, Astrid Cruaud 2025. Targeted hybridization capture for phylogenomics. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw12jzlmk/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: Working
We use this protocol and it's working
Created: August 08, 2024
Last Modified: November 07, 2025
Protocol Integer ID: 104962
Keywords: Illumina, Capture, Ultraconserved Elements, NEBNextUltraII, Probe, DNA Library, AMPure XP beads, myBaits, Daicel Arbor Biosciences, tagged adapters, Hymenoptera, insects, phylogeny, phylogenomics, parasitoid, enrichment, targeted hybridization capture for phylogenomics ultra, phylogenomics ultra, insects of the order hymenoptera, ultra ii dna library prep kit, phylogenetic relationship, order hymenoptera, targeted hybridization capture, genomic library construction, hybridization capture, insect, daicel arbor bioscience, samples with dna quantity, evolutionary history, specimen, genomic region, type specimen, dna quantity, small amounts of dna, dry specimen, gene discovery, input dna, specimens per reaction, dna
Abstract
Ultra-Conserved Elements (UCEs) are genomic regions that remain highly conserved across distantly related taxa. By sequencing both UCEs and their flanking regions, we can reconstruct evolutionary histories and investigate population-level relationships across a wide range of organisms. Hybridization capture, the core method used here, is versatile and can also be applied to genotyping, gene discovery, rare variant detection, indel and copy number variation analysis, as well as exome sequencing.
We developed this protocol to resolve phylogenetic relationships within the superfamily Chalcidoidea and, more broadly, to study insects of the order Hymenoptera.

Our protocol combines and optimizes two key steps, using commercial kits from NEB and Daicel Arbor Biosciences: 1) Genomic library construction from very small amounts of DNA. 2) Target enrichment to selectively capture libraries containing the UCEs of interest (with pools of 16 specimens per reaction).

We successfully applied this approach to both ethanol-preserved and dry specimens, ranging from 0.1 cm to several centimeters in size, including rare and historical museum material (type specimens).
DNA was extracted using the Qiagen’s DNeasy Blood & Tissue Kit. An average of 30 ng of input DNA was used for library preparation, but samples with DNA quantities below the Qubit detection limit were also successfully processed.
The protocol is scalable, enabling the processing of batches of up to 96 samples in parallel for sequencing on Illumina platforms (e.g., MiSeq, NovaSeq X+).
To minimize costs, we use half the reagents of an NEBNext Ultra II DNA Library Prep Kit, effectively doubling its capacity to produce 192 libraries per kit, and we streamlined several steps to reduce handling time.
For multiplexing, each library is uniquely indexed prior to hybridization capture. We designed 32 P1 adapters and 24 P2 adapters (Illumina-compatible), allowing up to 768 uniquely indexed libraries to be sequenced simultaneously.

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Materials
Equipment and consumables :

Note
All equipment and consumables described here correspond to those available in our laboratory, but they may be substituted with equivalent alternatives.

1. Pipette Tips (10µl - 200µl - 1000µl)
2. Single-Channel Pipettes (10µl - 200µl - 1000µl)
3. Multi-Channel Pipettes (10µl - 200µl)
4. Multipette E3/E3x electronic multi-dispenser pipette (Eppendorf)
5. Combitips advanced dispenser tips (Eppendorf)
6. Vortex
7. Plate and tube centrifuge (Eppendorf)
8. 0.65 ml Bioruptor‱ Pico Microtubes (Diagenode)
9. Tube holder for 0.65 ml tubes - Bioruptor‱ Pico (Diagenode)
10. Bioruptor‱ Pico (Diagenode)
11. Tube DNA LoBind 1,5 ml PCR clean (Eppendorf)
12. PCR plate without skirt, 96 well, transparent, High Profile, 200µl, PCR Performance Tested, PP (Sarstedt)
13. Invitrogen‱ DynaMag‱-2 Magnet (Invitrogen)
14. SPRIPlate 96R Ring Super Magnet Plate (Beckman Coulter)
15. Sealing film Adhesive PCR film 4TITUDE (Dutscher)
16. Adhesive Aluminium Film 131x83.6mm (Dutscher)
17. Thermocycler MasterCycler (Eppendorf)
18. Reaction tube, 1.5 ml, PP (Sarstedt)
19. Tube with conical bottom 15 ml (bulk) Falcon‱ (Dutscher)
20. Tube with conical bottom 50 ml (bulk) Falcon‱ (Dutscher)
21. Qubit assay tubes (Invitrogen)
22. Qubit 4 Fluorometer (Thermo Fisher)
23. Axygen‱ 0.2 mL Thin Wall PCR Tubes with Flat Cap, Clear, Nonsterile (Fisher)
24. ThermoMixer‱ C (Eppendorf)
25. Agilent 2100 Bioanalyzer (Agilent)
26. LightCycler‱480 (Roche)


Reagents :


Note
For each reagent, refer to the Safety Data Sheet (SDS) for information on proper handling and disposal.

1. NaCl (Merck)
2. Tris-Cl (Merck)
3. Buffer EB (Qiagen)
4. Ethanol absolute
5. AMPure XP Reagent (Beckman Coulter)
6. NEBNext‱ Ultra‱ II DNA Library Prep Kit for Illumina‱ (NEB E7645L)
7. Nuclease-Free Water (Qiagen)
8. Qubit dsDNA HS Assay Kit
9. myBaits UCE Hymenoptera 1.5Kv1 (Daicel Arbor Biosciences)
10. 2X KAPA HiFi HotStart ReadyMix (Roche)
11. High Sensitivity DNA Chip (Agilent)
12. Lib Quantification Kit Illumina/Universal (Roche)
Troubleshooting
Safety warnings
All these manipulations are performed with filter tips, dedicated pipettes and according to the principles of forward motion and good laboratory practice. Refer to the procedures of the laboratories used.
Before You Start :
Preparing tagged adapters :

Note
Homemade tagged adapters will be used during library preparation. Each tagged P1 and P2 adapter consists of two oligonucleotides (Forward and Reverse) hybridized together. The oligonucleotides were ordered from IDT (Integrated DNA Technologies). The sequences of all adapters are provided in the attached file "Sequences_adapters_P1andP2_protocols.io" available in the description tab of this protocol.

Prepare annealing buffer and store it at 4°C :

AB buffer 10X, Concentration500 millimolar (mM) NaCl , Concentration100 millimolar (mM) Tris-Cl , (pH 7.5-8.0)

Prepare the oligonucleotides at a concentration of 100µM :

1. Centrifuge the lyophilized oligo tubes thoroughly
2. Resuspend the lyophilized oligos at Concentration100 micromolar (µM) in ultrapure H2O by adding the volume of H2O indicated on the sheet provided by IDT,
3. Incubate the tubes at Temperature4 °C overnight,
4. The next day, vortex and centrifuge the tubes thoroughly before proceeding to quantification.


Prepare each oligonucleotide pair at a final concentration of 25 µM :

1. Vortex and centrifuge the resuspended 100µM oligos thoroughly,
2. Pool the oligo pairs (Forward/Reverse) into 1.5 ml non-stick tubes as follows:

Amount25 µL of Forward oligo (100µM)
Amount25 µL of Reverse oligo (100µM)
Amount10 µL of AB Buffer (10X)
Amount40 µL of ultra pure H2O

3. Vortex and centrifuge the pools thoroughly, then distribute Amount50 µL of each pool into 2 × 0.2 ml tubes or into a PCR plate,
4. Close the tubes tightly or seal the plate with PCR-specific film.
Hybridization of oligonucleotide pairs to generate adapters :

1. Place the tubes or plate in a thermal cycler, with the heated lid set to ≥ 75°C, and run the following program :

Duration00:02:00 at Temperature95 °C
Duration00:01:00 at Temperature95 °C (15 cycles with -1.7°C / cycle)
Duration00:01:00 at Temperature69 °C (40 cycles with -1°C / cycle)
Hold at Temperature10 °C

2. Vortex and centrifuge the tubes or plate containing the hybridized oligos,
3. Check the concentration of the adapters by measuring Amount2 µL of a 1:15 dilution using the Qubit‱ dsDNA HS Assay kit, following the manufacturer’s instructions.
Adjustment of adapter concentrations to 10 µM :

1. Dilute the AB buffer 10 times (1:10) in ultrapure water to obtain 1X AB buffer,
2. Adjust adapter concentration to Concentration10 micromolar (µM) in 1X AB buffer, with a final volume of Amount100 µL . The AB buffer prevents the dissociation of the two strands of the adapters.
3. Vortex and centrifuge the adapters thoroughly, then aliquot them into 0.2 mL tubes or tube strips for storage at Concentration10 micromolar (µM) .
4. Store the aliquots at Temperature-20 °C ,
5. Adapters will be used at Concentration1.5 micromolar (µM) in this protocol.

Part I : Library preparation (using the NEBNext® Ultra™ II DNA Library Prep Kit)
Normalize DNA for DNA shearing :

Prepare Amount100 µL of each 96 DNA samples in 96 × 0.65 mL microtubes (Diagenode). An average of 30 ng of input DNA is used for library preparation, but samples with DNA quantities below the Qubit detection limit were also successfully processed.

Note
The microtubes can be stored at Temperature4 °C overnight or at Temperature-20 °C for up to one month before shearing.

DNA Shearing (using the Diagenode Bioruptor Pico) :

1. Keep the microtubes at Temperature4 °C or on ice throughout the shearing step,
2. Briefly vortex and centrifuge the microtubes,
3. Remove any bubbles you may see in the microtubes,
4. Install the microtubes in the 0.5/0.65ml tube holder,
5. To obtain ~400 bp fragments, select the following program: 15 s ON / 90 s OFF for 8 cycles.
6. Keep the microtubes at Temperature4 °C or on ice after shearing,

Note
The sheared DNA can be stored at Temperature4 °C overnight or atTemperature-20 °C for up to one month before the next step.


DNA Reconcentration (optional) :

Note
For samples with more than 10 ng of input DNA for shearing, we use half of the shearing volume (50 µL) as input for the next step (End Prep). For samples with less than 10 ng of input DNA, we reconcentrate the entire DNA volume to 50 µL before proceeding. Therefore, the step detailed below is only required for certain samples.

1. Let the Beckman Coulter‱ Agencourt AMPure XP beads stand for Duration00:30:00 at TemperatureRoom temperature
2. Bring the fragmented DNA microtubes to TemperatureRoom temperature
3. Vortex the beads thouroughtly,
4. Transfer the Amount100 µL of fragmented DNA into 1.5mL DNA LoBind tubes or into the wells of a 96-well plate,
5. Add Amount100 µL of Ampure beads to reach a final volume of Amount200 µL ,
6. Mix thoroughly by pipetting,
7. Incubate for Duration00:05:00 at TemperatureRoom temperature ,
8. Prepare fresh 70% EtOH (for 96 tubes: mix Amount30 mL of EtOHabs + Amount13.8 mL of H2OmiliQ),
9. Move tubes or plate into a magnet rack for Duration00:05:00 or until the supernatant appear clear,
10. Remove and discard the supernatant without disturbing beads,
11. Add Amount200 µL of 70% fresh EtOH into the tubes or plate without removing the tubes or plate from the magnetic rack ,
12. Incubate for Duration00:00:30 at TemperatureRoom temperature ,
13. Remove and discard EtOH,
14. Repeat steps 10 to 12 for a total of two washes,
15. Dry tubes or plate with beads for Duration00:08:00 at TemperatureRoom temperature to ensure all traces of EtOH are removed. WARNING : Do not over-dry the beads, as this will significantly decrease elution efficiency,
16. Add Amount50 µL of EB Buffer (Qiagen) without removing the tubes or plate from the magnetic rack,
17. Remove tubes or plate from magnetic rack and mix well by pipetting,
18. Incubate for Duration00:02:00 at TemperatureRoom temperature ,
19. Move tubes or plate into a magnet rack for Duration00:05:00 or until the supernatant appear clear,
20. Transfer Amount50 µL of supernatant into wells of a 96-well plate for the next step.

Note
For samples where fragmented DNA did not require reconcentration, transfer Amount50 µL of DNA in the plate and keep the remaining Amount50 µL as a back-up (store it at Temperature-20 °C ).


End Prep :

1. Thaw NEBNext Ultra II End Prep Reaction Buffer and keep NEBNext Ultra II End Prep Enzyme Mix TemperatureOn ice ,
2. Briefly vortex and centrifuge the NEBNext Ultra II End Prep Reaction Buffer,
3. Prepare the End Prep mix for 96 samples (a volume for 100 samples is calculated here to ensure sufficient volume is available) :

Amount350 µL of NEBNext Ultra II End Prep Reaction Buffer (or Amount3.5 µL per sample)
Amount150 µL of NEBNext Ultra II End Prep Enzyme Mix (or Amount1.5 µL per sample)

4. Vortex thoroughly and centrifuge the mix,
5. Add Amount5 µL of End Prep Mix in each well of the 96-well plate containing fragmented DNA,
6. Add Amount5 µL of EB Buffer (Qiagen) in each well of the 96-well plate to reach a final volume of Amount60 µL ,
7. Seal the plate with PCR-specific foil,
8. Briefly vortex and centrifuge the plate,
9. Place the plate in a thermal cycler with the heated lid set to ≥ 75°C, and run the following program :

Duration00:30:00 at Temperature20 °C
Duration00:30:00 at Temperature65 °C
Hold at Temperature10 °C

Note
If necessary, samples can be stored at Temperature-20 °C ; however, a slight loss in yield (~20%) may be observed. NEB recommends proceeding with adapter ligation before stopping.



Adapters Ligation :

Note
In this protocol, we use homemade P1 and P2 adapters (compatible with Illumina sequencing) that are not included in the NEBNext® Ultra™ II DNA Library Prep Kit. To prepare them, refer to the "Before You Start" section of this protocol.

1. Let the Beckman Coulter‱ Agencourt AMPure XP beads stand for Duration00:30:00 at TemperatureRoom temperature ,
2. Thaw the Concentration1.5 micromolar (µM) P1 and P2 adapters at TemperatureRoom temperature , then briefly vortex and centrifuge before use,
3. Thaw the NEBNext Ultra II Ligation Master Mix. Gently vortex and centrifuge before use,
4. Thaw the NEBNext Ultra II Ligation Enhancer. Vortex and centrifuge before use,
5. Keep the reagents on a Temperature0 °C cooling rack during mix preparation.
6. Prepare the Ligation mix for 96 samples (a volume for 100 samples is calculated here to ensure sufficient volume is available) :

Amount1500 µL of NEBNext Ultra II Ligation master mix (or Amount15 µL per sample)
Amount50 µL of NEBNext Ultra II enhancer (or Amount0.5 µL per sample)

7. Gently vortex and centrifuge the mix, then keep it on theTemperature0 °C cooling rack.
8. Remove the plate containing Amount60 µL of DNA (from the end prep step) from the thermocycler and place it on a Temperature0 °C rack,
9. Add Amount15.5 µL of Ligation mix in each well of the 96-well plate. Note that the mix is very viscous,
10. Add Amount15.5 µL of EB Buffer (Qiagen) in each well of the 96-well plate to reach a final volume of Amount91 µL ,
11. Add Amount1.25 µL of P1 adapters (1.5µM) and Amount1.25 µL of P2 adapters (1.5 µM) to each well (final reaction volume = 93.5 µL).

For 96 libraries, we use the following layout :


12. Seal the plate with aluminium foil,
13. Briefly vortex and centrifuge the plate,
14. Place in a thermal cycler, without heated lid, and run the following program :

Duration00:15:00 at Temperature20 °C
Hold at Temperature10 °C

Note
If bead purification cannot be performed immediately, store the samples at Temperature-20 °C (e.g., during lunch break or overnight).


Cleanup of Adapter-ligated DNA :

1. Let the Beckman Coulter‱ Agencourt AMPure XP beads stand for Duration00:30:00 at TemperatureRoom temperature ,
2. If needed thaw and centrifuge the plate containing the ligated DNA,
3. Vortex the beads thoroughly,
4. Transfer Amount93.5 µL of ligated DNA into 0.2 mL PCR tube strips or into a 96-well plate,
5. Add Amount75 µL (0.8X) of Ampure beads to reach a final volume of Amount168.5 µL ,
6. Mix thoroughly by pipetting (or vortexing),
7. Incubate for Duration00:05:00 at TemperatureRoom temperature ,
8. Prepare fresh 70% EtOH (for 96 tubes: Amount30 mL of EtOHabs + Amount13.8 mL of H2OmiliQ),
9. Move tubes or plate into a magnet rack for Duration00:05:00 or until the supernatant appear clear,
10. Remove and discard the supernatant without disturbing beads,
11. Add Amount200 µL of 70% fresh EtOH into the tubes or plate without removing the tubes or plate from the magnetic rack ,
12. Incubate for Duration00:00:30 at TemperatureRoom temperature ,
13. Remove and discard EtOH,
14. Repeat steps 11 to 13 for a total of two washes,
15. Dry tubes or plate with beads for Duration00:08:00 at TemperatureRoom temperature to ensure all traces of EtOH are removed. WARNING : Do not over dry the beads as this will significantly decrease elution efficiency,
16. Add Amount15 µL of EB Buffer (Qiagen) without removing the tubes or plate from the magnetic rack,
17. Remove tubes or plate from magnetic rack and mix well by pipetting,
18. Incubate for Duration00:02:00 at TemperatureRoom temperature ,
19. Move tubes or plate into a magnet rack for Duration00:05:00 or until the supernatant appear clear,
20. Transfer Amount15 µL of supernatant in a 96-well plate for the next step.

Note
If necessary, the plate can be stored at Temperature-20 °C

PCR Enrichment of Adapter-ligated DNA :

Note
For the enrichment PCR, we use 10µM primers compatible with the adapters used previously.

Forward    5'-AATGATACGGCGACCACCGA-3'
Reverse    5'-CAAGCAGAAGACGGCATACGA-3'

1. Thaw the NEBNext Ultra II Q5 master mix. Do not vortex, but gently centrifuge before use,
2. If needed, thaw and centrifuge the plate containing the purified Adapter-ligated DNA,
3. If needed, thaw, vortex and centrifuge the PCR primers Forward and Reverse Concentration10 micromolar (µM)
4. Prepare the Enrichment PCR mix for 96 samples (a volume for 100 samples is calculated here to ensure sufficient volume is available) :

Amount1250 µL of NEBNext Ultra II Q5 master mix (or Amount12.5 µL per sample)
Amount100 µL of Primer Forward 10µM (or Amount1 µL per sample)
Amount100 µL of Primer Reverse 10µM (or Amount1 µL per sample)

5. Gently vortex and centrifuge the mix,
6. Add Amount14.5 µL of Enrichment PCR mix to each well of the 96-well plate that contains the purified Adapter-ligated DNA,
7. Then, add Amount14.5 µL of EB Buffer (Qiagen) to each well of the 96-well plate to reach a final volume of Amount44 µL .
8. Seal the plate with PCR-specific foil,
9. Briefly vortex and centrifuge the plate,
10. Place the plate in a thermal cycler, with the heated lid set to ≥ 75°C, and run the following program :

Duration00:00:30 at Temperature98 °C
Followed by 15 cycles :
Duration00:00:10 at Temperature98 °C
Duration00:01:15 at Temperature65 °C
And a final extension :
Duration00:05:00 at Temperature65 °C
Hold at Temperature10 °C

Note
If necessary, samples can be stored at Temperature-20 °C

Quantification of the amplified libraries :

1. If needed, thaw the amplified libraries, vortex and centrifuge the plate,
2. Quantify Amount2 µL from each well using the Qubit‱ dsDNA HS Assay kit, following the manufacturer’s instructions.
Equimolar pooling and bead-based re-concentration of amplified libraries :

Note
At this stage, the libraries (one per sample) could be sequenced. However, to enrich them for fragments containing only UCEs, a probe hybridization capture step will be performed.
Before capture, the libraries will be pooled in groups of 16 (100 ng per sample) to generate a total of six pools.

1. Let the Beckman Coulter‱ Agencourt AMPure XP beads stand for Duration00:30:00 at TemperatureRoom temperature ,
2. Pool the libraries in groups of 16 (100 ng each) into six 1.5 mL Eppendorf Low-Bind tubes, for example following the plate layout below:

Pool 1 = columns 1 & 2
Pool 2 = columns 3 & 4
...
Pool 6 = columns 11 & 12


3. Measure the exact volume of each pool,
4. Vortex the beads thoroughly,
5. Add Amount0.8 V of Ampure beads for Amount1 V of libraries pool,
6. Mix thoroughly by pipetting or vortexing,
7. Incubate for Duration00:05:00 at TemperatureRoom temperature ,
8. Prepare fresh 70% EtOH (for 6 tubes: Amount2 mL of EtOHabs + Amount920 µL of H2OmiliQ),
9. Move tubes into a magnet rack for Duration00:05:00 or until the supernatant appear clear,
10. Remove and discard the supernatant without disturbing beads,
11. Add Amount200 µL of 70% fresh EtOH into the tubes without removing them from the magnetic rack ,
12. Incubate for Duration00:00:30 at TemperatureRoom temperature ,
13. Remove and discard EtOH,
14. Repeat steps 11 to 13 for a total of two washes,
15. Dry tubes with beads for Duration00:08:00 at TemperatureRoom temperature to ensure all traces of EtOH are removed. WARNING : Do not over dry the beads as this will significantly decrease elution efficiency,
16. Add Amount10 µL of EB Buffer (Qiagen) without removing the tubes from the magnetic rack,
17. Remove the tubes from the magnetic rack and mix thoroughly by pipetting,
18. Incubate for Duration00:02:00 at TemperatureRoom temperature ,
19. Move tubes back to the magnet rack for Duration00:05:00 or until the supernatant appear clear,
20. Transfer Amount10 µL of supernatant into new, unused Eppendorf Low-Bind 1.5 ml tubes,
21. Briefly vortex and centrifuge the tubes,
22. Quantify Amount1 µL of each pool using the Qubit‱ dsDNA HS Assay kit, following the manufacturer’s instructions.
23. In 0.2 mL low-bind tubes (here labeled "LIB 1" to "LIB 6"), normalize 7 µl of each pool to obtain 100-500 ng of material for the probe hybridization capture protocol.

Note
If necessary, samples can be stored atTemperature4 °C overnight or at Temperature-20 °C for up to 1 month.

At this stage, 96 individual genomic DNA libraries have been constructed, tagged, and pooled into six groups of 16. Using a commercial kit from Daicel Arbor Biosciences (myBaits UCE Hymenoptera 1.5Kv1), each pool will now be enriched for the target sequences (UCEs). This kit contains complementary oligonucleotides to the UCEs, transcribed into biotinylated RNA, which serve as probes for hybridization. The baits to capture UCEs were designed by Faircloth et al. 2015. doi: 10.1111/1755-0998.12328


Part II : Probe hybridization capture of libraries (using the myBaits® hybridization capture kit)
Hybridization :

Note
The protocol described here follows the recommendations of version v5.03 (June 2023) of the Hybridization Capture for Targeted NGS Manual from Daicel Arbor Biosciences. Please ensure compatibility with the version of the kit you are using. If necessary, the most recent version can be downloaded from the manufacturer’s website.

In this step, sequencing libraries are mixed with various blocking nucleic acids, denatured, and then combined with a mixture of hybridization reagents (including baits). The hybridization reactions are then incubated for several hours to allow the baits to encounter and hybridize with the target UCEs.

1. Preheat a thermomixer to Temperature60 °C ,
2. Thaw the following reagents and keep them at TemperatureRoom temperature . Vortex and centrifuge before use. If HybN and/or HybS remain cloudy, place them at Temperature60 °C for Duration00:05:00 until the crystals are completely dissolved :
Hyb N
Hyb S Hyb D
Hyb R
Block C
Block O
Block X
3. Thaw the baits and keep them TemperatureOn ice
4. Prepare the hybridization mix for 6 pools :

Amount55.5 µL of Hyb N (or Amount9.25 µL per pool)
Amount21 µL of Hyb D (or Amount3.5 µL per pool)
Amount3 µL of Hyb S (or Amount0.5 µL per pool)
Amount7.5 µL of Hyb R (or Amount1.25 µL per pool)
Amount33 µL of Baits (or Amount5.5 µL per pool)

5. Gently vortex and centrifuge the mix,
6. Incubate the mix at Temperature60 °C for Duration00:10:00 in the thermomixer. Remove the mixture from the heat block and allow it to return to TemperatureRoom temperature for Duration00:05:00 ,
7. For each capture reaction (6 in total here), aliquot Amount18.5 µL of Hybridization mix into 0.2 mL low-bind tubes (here labeled "HYB 1" to "HYB 6"),
8. Prepare the blockers mix for 6 pools :

Amount15 µL of Block O (or Amount2.5 µL per pool)
Amount15 µL of Block C (or Amount2.5 µL per pool)
Amount3 µL of Block X (or Amount0.5 µL per pool)

9. Gently vortex and centrifuge the mix,
10. Thaw the tubes containing Amount7 µL of each previously prepared library pool (here "LIB 1" to "LIB 6"),
11. Add Amount5 µL of blockers mix to each "LIB" tubes and mix by pipetting (total volume Amount12 µL ),
12. Preheat a thermocycler, with the heated lid set to ≥ 75°C, and program it with the following thermal cycling conditions :

Duration00:05:00 at Temperature95 °C (step 1)
Duration00:05:00 at Temperature65 °C (step 2)
Hold at Temperature65 °C

13. Put the "LIB" tubes in the thermal cycler, close the lid, and start the program,


14. Once the cycler reaches the hybridization temperature (65°C, step 2), pause the program, put the "HYB" tubes in the thermocycler, close the lid, and resume the program,


15. Once step 2 of the program is complete, and while keeping all tubes in the thermocycler, pipette Amount18 µL from each "HYB" tubes into the corresponding "LIB" tubes. Use a multichannel pipette to facilitate the process. Gently homogenize by pipetting up and down 5 times,


16. Dispose of the "HYB" tubes, close the lid, and allow the reactions to incubate at the hybridization temperature (Temperature65 °C ) for Duration16:00:00 to Duration24:00:00 ,

Note
WARNING: The process must NOT be stopped here — it is essential to proceed immediately to the next step.





Bind and wash :

Note
In this step, bait–target hybrids are bound to streptavidin-coated magnetic beads, and most non-target DNA is removed through several rounds of washing with pre-warmed buffer. This procedure is typically performed the day after completion of the hybridization step.

Preparation of Wash Buffer X:

1. Preheat a thermomixer to Temperature65 °C ,
2. Thaw the following reagents and keep them at TemperatureRoom temperature . Vortex and centrifuge before use (if HybS remain cloudy, place it at Temperature60 °C for Duration00:05:00 until the crystals are completely dissolved) :
Hyb S Binding Buffer
Wash Buffer
Streptavidin Beads
Buffer E
3. In a 15 ml tube, prepare Wash Buffer X for 6 pools :

Amount54 µL of Hyb S (or Amount9 µL per pool)
Amount5400 µL of Ultra Pure H20 (or Amount900 µL per pool)
Amount1362 µL of Wash Buffer (or Amount227 µL per pool)

4. Aliquot Wash Buffer X into 6 x 1.5 mL tubes and incubate at Temperature65 °C for Duration00:30:00 before use.


Preparation of Streptavidin-Coated Magnetic Beads :

1. For each capture reaction (6 here), aliquot Amount30 µL of beads into 1.5mL Eppendorf Low-Bind tubes (6 here),
2. Pellet the beads on a magnetic rack for Duration00:02:00 until the supernatant appear clear. While keeping the tubes on the magnet, carefully remove and discard the supernatant,
3. Add Amount200 µL of Binding Buffer to each bead aliquot. Remove the tubes from the magnet, vortex to resuspend the beads and centrifuge briefly. Place tubes into the magnetic rack and pellet the beads; remove and discard the supernatant,
4. Repeat step 3 above twice for a total of three washes,
5. Resuspend each washed bead aliquot in Amount70 µL of Binding Buffer.

Binding of Bait–Target Hybrids to Streptavidin-Coated Beads :

1. Heat the bead aliquots in a thermomixer at Temperature65 °C for at least Duration00:02:00
2. Transfer each capture reaction to the heated bead aliquots. Mix by pipetting,
3. Incubate the tubes containing libraries and beads on the thermomixer at Temperature65 °C for Duration00:05:00 . Agitate at the 2.5 minute mark by flicking or inverting the tubes to keep the beads suspended, followed by briefly centrifuging.

Washing of Beads :

1. Pellet the beads into a magnetic rack for Duration00:02:00 until the supernatant appear clear. While keeping the tubes on the magnet, remove and discard the supernatant,
2. Add Amount375 µL of of warmed Wash Buffer X to the beads, remove tubes from magnetic rack and briefly vortex them,
3. Place the tubes immediately in a thermomixer and incubate for Duration00:05:00 at Temperature65 °C (withDuration00:00:10 of shaking every Duration00:02:00 at 900 rpm),
4. Pellet the beads into a magnetic rack for Duration00:02:00 until the supernatant appear clear. While keeping the tubes on the magnet, remove and discard the supernatant,
5. Repeat step 2 to 4 twice for a total of three washes,
6. After the final pelleting step, carefully remove as much liquid as possible without disturbing the bead pellet.

Resuspension and Amplification of Libraries :

1. Thaw the 2X KAPA HiFi HotStart Ready Mix at TemperatureRoom temperature ,
2. Thaw the Concentration10 micromolar (µM) forward and reverse PCR primers (See Part I Section 8) at TemperatureRoom temperature
3. Add Amount30 µL of Buffer E to the libraries+beads from the previous step and thoroughly resuspend the beads by pipetting,

Note
The beads can be kept resuspended in Buffer E at Temperature4 °C until the next day or at Temperature-20 °C for up to one month before proceeding to the next step.

4. Transfer Amount15 µL of each library+bead resuspension twice into a PCR plate. The goal is to perform two PCR reactions for each library+bead pool to increase the diversity of fragments used during amplification,
5. Prepare the amplification mix for 12 samples (6 library+bead resuspension x2), a volume for 13 samples is calculated here to ensure sufficient volume is available :

Amount325 µL of 2X KAPA HiFi HotStart Ready Mix (or Amount25 µL per sample)
Amount65 µL of Ultra Pure H20 (or Amount5 µL per sample)
Amount32.5 µL of Primer Forward 10µM (or Amount2.5 µL per sample)
Amount32.5 µL of Primer Reverse 10µM (or Amount2.5 µL per sample)

6. Gently vortex and centrifuge the mix,
7. Add Amount35 µL of amplification mix to each well of the plate containing Amount15 µL of library-bead resuspension,
8. Seal the plate with PCR-specific foil,
9. Briefly vortex and centrifuge the plate,
10. Place the plate in a thermal cycler with the heated lid set to ≥ 75°C, and run the following program :

Duration00:02:00 at Temperature98 °C
Followed by 14 cycles :
Duration00:00:20 at Temperature98 °C
Duration00:00:30 at Temperature65 °C
Duration00:00:45 at Temperature72 °C
And a final extension :
Duration00:05:00 at Temperature72 °C
Hold at Temperature10 °C

PCR clean-up using AMPure XP beads :

Note
Before performing the PCR clean-up, the streptavidin beads must be removed as detailed below.

1. Let the Beckman Coulter‱ Agencourt AMPure XP beads stand for Duration00:30:00 at TemperatureRoom temperature ,
2. Place the PCR plate on a magnetic rack for Duration00:05:00 to pellet the streptavidin beads,
3. DO NOT DISCARD THE SUPERNATANT, instead, transfer and pool the supernatants (library by library) into 1.5 mL low-bind tubes. Here, since two 50 µL enrichment PCR reactions were performed per library, a total of 100 µL of supernatant is recovered for each library.
4. Vortex the AMPure beads thoroughly,
5. Add Amount100 µL of AMPure Beads (1X) in each tubes containing Amount100 µL of PCR products to reach a final volume of Amount200 µL ,
6. Mix well by pipetting or vortexing,
7. Incubate for Duration00:05:00 at TemperatureRoom temperature ,
8. Prepare fresh 70% EtOH (for 6 tubes: Amount2 mL of EtOHabs + Amount920 µL of H2OmiliQ),
9. Move tubes into a magnetic rack for Duration00:05:00 or until the supernatant appears clear,
10. Remove and discard the supernatant without disturbing the beads,
11. Add Amount200 µL of 70% fresh EtOH into the tubes without removing them from the magnetic rack ,
12. Incubate for Duration00:00:30 at TemperatureRoom temperature ,
13. Remove and discard EtOH,
14. Repeat steps 12 to 14 for a total of two washes,
15. Dry tubes with beads for Duration00:08:00 at TemperatureRoom temperature to ensure all traces of EtOH are removed. WARNING : Do not over dry the beads as this will significantly decrease elution efficiency,
16. Add Amount25 µL of EB Buffer (Qiagen) without removing the tubes from the magnetic rack,
17. Remove tubes from magnetic rack and mix well by pipetting or vortexing,
18. Incubate for Duration00:02:00 at TemperatureRoom temperature ,
19. Move tubes back to the magnetic rack for Duration00:05:00 or until the supernatant appears clear,
20. Transfer Amount25 µL of supernatant in new, unused Eppendorf Low-Bind 1.5 ml tubes.
21. Quantify Amount2 µL of each library using the Qubit‱ dsDNA HS Assay kit, following the manufacturer’s instructions.

Library Quality and Quantity Assessment :
Quality control by microfluidic electrophoresis :

1. Take the reagents from the Agilent High Sensitivity DNA kit out of the fridge 30 minutes before use,
2. Load 1 µl of undiluted libraries onto an High Sensitivity DNA Chip and run it in a Agilent 2100 Bioanalyzer, following the supplier's recommendations.
3. An average library size of 520 bp is expected (400 bp insert + 120 bp adapters).

Note
Microfluidic electrophoresis separates NGS library fragments based on size, enabling validation of the construct size and verification of the absence of short fragments (e.g., adapter dimers). This technique also provides an approximate estimate of library concentration. However, a more accurate quantification method must be used before performing equimolar pooling of NGS libraries for sequencing.

Quantity Control by qPCR :

Note
Before NGS libraries are loaded onto the sequencer, they must be quantified and normalized to ensure that each library is sequenced to the desired depth and yields the required number of reads.
Quantification by qPCR is performed by amplifying DNA fragments containing the P1 and P2 adapters. Because the PCR primers are designed to anneal specifically to the adapter sequences, the qPCR assay detects only properly adapted, amplifiable libraries capable of cluster formation during sequencing. However, qPCR can also amplify adapter dimers. Therefore, it is critical to verify their absence by microfluidic electrophoresis (e.g., Bioanalyzer) prior to quantification.

This step is carried out using the 'Lib Quantification Kit Illumina/Universal' commercialized by Roche on a LightCycler‱ 480, following the manufacturer’s instructions.

The final library concentration is calculated using the following formula : Final library concentration = [Length of qPCR DNA standard fragments (bp) / Average lengths of library fragments (bp)] x Library concentration determined by qPCR

Note that In the 'Lib Quantification Kit Illumina/Universal', Length of qPCR DNA standard fragments = 452bp
Equimolar pooling of libraries for Illumina sequencing :

Based on the qPCR quantification results, mix the libraries equimolarly according to the requirements of the Illumina sequencer to be used.
Protocol references
The baits used in this protocol were designed by Faircloth et al. 2015.
Faircloth, B.C., Branstetter, M.G., White, N.D., S.G., B., 2015. Target enrichment of ultraconserved elements from arthropods provides a genomic perspective on relationships among Hymenoptera. Mol. Ecol. Resour. 15, 489-501, https://doi.org/10.1111/1755-0998.12328.

We used this protocol to capture UCEs from (micro-)hymenoptera but also moths (with another set of baits) :
See for example :
#Cruaud, A., Rasplus, J.-Y., Zhang, J., Burks, R., Delvare, G., Fusu, L., Gumovsky, A., Huber, J.T., Janšta, P., Mitroiu, M.-D., Noyes, J.S., van Noort, S., Baker, A., Böhmová, J., Baur, H., Blaimer, B.B., Brady1, S.G., Bubeníková, K., Chartois, M., Copeland, R.S., Dale-Skey Papilloud, N., Dal Molin, A., Dominguez, C., Gebiola, M., Guerrieri, E., Kresslein, R.L., Krogmann, L., Moriarty Lemmon, E., Murray, E., Nidelet, S., Nieves Aldrey, J.L., Perry, R., Peters, R.S., Polaszek, A., Sauné, L., Torréns, J., Triapitsyn, S., Tselikh, E.V., Yoder, M., Lemmon, A., Woolley, J.B., Heraty, J.M., 2024. The Chalcidoidea bush of life - Evolutionary history of a massive radiation of minute wasps. Cladistics 40, 34-63, https://doi.org/10.1111/cla.12561.
#Juvé, Y., Weyna, A., Lauroua, E., Nidelet, S., Khaldi, M., Barech, G., Lebas, C., Rasplus, J.Y., Cruaud, A., Condamine, F.L., Romiguier, J., 2025. Phylogenomics of Messor harvester ants (Hymenoptera: Formicidae: Stenammini) unravels their biogeographical origin and diversification patterns. Syst. Entomol. 50, 1025-1040, https://doi.org/10.1111/syen.12693.
#Rougerie, R., Cruaud, A., Arnal, P., Ballesteros-Mejia, L., Condamine, F.L., Decaëns, T., Elias, M., Gey, D., Hebert, P.D.N., Kitching, I.J., Lavergne, S., Lopez-Vaamonde, C., Murienne, J., Cuenot, Y., Nidelet, S., Rasplus, J.-Y., under review. Phylogenomics Illuminates the Evolutionary History of Wild Silkmoths in Space and Time (Lepidoptera: Saturniidae). https://doi.org/10.1101/2022.03.29.486224.
Acknowledgements
This work was supported by government funding managed by the French National Research Agency (ANR) under the France 2030 plan (EcoControl project, reference ANR-24-PEAE-0004, within the PEPR Agroecology and Digital).