Apr 04, 2025
Version 1
Bravo workstation: automated indexing, purification and quantification of DNA libraries V.1
- 1Max Planck Institute for Evolutionary Anthropology;
- 2Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, D-04103 Leipzig, Germany
- MPI EVA Ancient DNA Core Unit
Protocol Citation: Sarah Nagel, Ayinuer Aximu Petri, Merlin Szymanski, Elena Essel, Matthias Meyer 2025. Bravo workstation: automated indexing, purification and quantification of DNA libraries. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g71w48gwz/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: July 31, 2024
Last Modified: April 04, 2025
Protocol Integer ID: 104355
Funders Acknowledgements:
Max Planck Society
Grant ID: -
Abstract
We here provide a protocol for the simultaneous amplification and dual-indexing of 96 DNA libraries through automated liquid handling using the Bravo NGS workstation (Agilent Technologies). The benefits of dual-indexing for preventing and detecting cross-contamination have been documented elsewhere (Kircher et al. 2012, Zavala et al. 2022). This protocol is optimized specifically for ancient and degraded DNA (i.e., libraries with short inserts) and is compatible with both double- and single-stranded methods for preparing Illumina-type libraries (e.g., Meyer and Kircher 2010, Gansauge et al. 2020). Amplification is carried out to PCR plateau, ensuring approximately equal yields across all samples. The use of AccuPrime Pfx polymerase minimizes length and GC biases during amplification (Dabney et al. 2012). Additionally, we provide sequences for 384 unique 8-bp indices, incorporated into the interior of both the P5 and P7 indexing primers.
The protocol also includes steps for purifying indexed libraries using Solid Phase Reversible Immobilization (SPRI) technology (deAngelis et al. 1995) and for determining the concentration of purified indexed libraries using a NanoDrop photospectrometer.
Some of the instructions, for example regarding the documentation and location of files, are specific to the environment and workflows of the Ancient DNA Core Unit of the MPI-EVA and have to be amended in other environments.
Notes
Default input volume of library into indexing PCR is 49 µl (the final library volume in library preparation with the ssDNA2.0 method), for a total reaction volume of 100 µl. However, lower input volumes are possible.
Input files are provided containing index combinations and Bravo pipetting commands for 384-well PCR plates, making for a total of 36,864 libraries that can be indexed with unique combinations of P7 and P5 indices with fast, column-wise pipetting. There is no overlap in P5 or P7 indices among four consecutive plates (labeled A, B, C and D), which together form one set of plates. Thus, four PCR plates can be set up consecutively using only a single P5 and P7 index primer plate each. Note that for half of the index combinations provided, the P5 index primer plate has to be rotated by 180° before insertion (indicated in the file name).
References
Dabney, J., & Meyer, M. (2012). Length and GC-biases during sequencing library amplification: A comparison of various polymerase-buffer systems with ancient and modern DNA sequencing libraries. BioTechniques, 52(2), 87-94.
DeAngelis, M. M., Wang, D. G., Hawkins, T. L. (1995) Solid-phase reversible immobilization for the isolation of PCR products. Nucleic Acids Res. 23(22):4742-3. doi: 10.1093/nar/23.22.4742.
Gansauge, M.-T., Aximu-Petri, A., Nagel, S., & Meyer, M. (2020). 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. Nature Protocols, 15, 2279-2300.
Kircher, M., Sawyer, S. & Meyer, M. (2012) Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform. Nucleic Acids Res., 40(1):e3, doi: 10.1093/nar/gkr771.
Meyer, M., & Kircher, M. (2010). Illumina sequencing library preparation for highly multiplexed target capture and sequencing. Cold Spring Harbor Protocols, 2010(6): prot5448.
Zavala, E. I., Ayinuer-Petri, A., Richter, J., Nickel, B., Vernot, B., & Meyer, M. (2022). Quantifying and reducing cross‐contamination in single‐ and multiplex hybridization capture of ancient DNA. Molecular Ecology Resources, 22(6), 2196-2207.
Materials
Materials
| Reagents/consumables | Supplier | Cat. no. | |
| Large-volume buffers/reagents | |||
| 80% EtOH (v/v) ‡ | self | - | |
| 33 % SPRI beads † | self | - | |
| TE buffer † | self | - | |
| Reagents | |||
| water | Sigma/Merck | 1153332500 | |
| 10x AccuPrime Pfx buffer | ThermoFisher Scientific | 12344032 | |
| AccuPrime Pfx DNA Polymerase | ThermoFisher Scientific | 12344032 | |
| P5 primer plate 384 * | self | - | |
| P7 primer plate 384 * | self | - | |
| Consumables | |||
| Bravo 96LT 250 µl sterile, filtered tips | Agilent | 19477-022 | |
| Bravo 96LT 250 µl sterile, filtered tips, empty boxes | Agilent | 19477-022 | |
| 5 ml Eppendorf tubes | Kisker | G05-ML | |
| NUNC 96 deepwell plate | NUNC | 736-0600 | |
| Peelable heatseal foil | Bio-Rad | 1814045 | |
| Twin.tec PCR Plate 96, semiskirted | neoLab Migge | VB-0398 | |
| Filter tip, 1000 μl, Diamond tips | neoLab Migge | CF-0425 | |
| Filter tip, 200 μl, Diamond tips | neoLab Migge | CF-0438 | |
| Filter tip, 10 μl, Diamond tips | neoLab Migge | CF-0417 | |
| HTS deep well reservoirs | Kisker | 97813 | |
| HTS low well reservoirs | Kisker | 97817 | |
| 96 Deep well PP Sqr well plate | Axygen | P-2ML-SQ-C-S | |
| Twin.tec PCR Plates 96, skirted | neoLab Migge | VB-0403 | |
† See documents in the Appendix for preparation of reagents, buffers and SPRI beads.
‡ Dilute 100% EtOH in water to obtain a 80 % solution.
* Order P5 and P7 indexing primers at 40 nmol synthesis scale (Eurogentec, RP-cartridge purification). See Appendix for the document describing the preparation of 384-well primer aliquot plates.
Equipment
- Bravo-B NGS workstation G5522A with 96-channel LT pipette head and associated equipment (red thermal plate insert for 96-well PCR plates, silver thermal plate insert for 96-well Nunc deep-well plates, two silver thermal plate insert for 384-well PCR plates, 96-well magnetic rack) for indexing library preparation
- Bravo-B NGS workstation G5522A with 96-channel LT pipette head and associated equipment (two red thermal plate insert for 96-well PCR plates, 96-well magnetic rack) for indexing library purification
- Hood for NGS workstation B with HEPA-filtered ventilation system and strip curtain (custom-built, optional)
- Centrifuge for PCR plates (e.g., Eppendorf, cat. no. 5948000913)
- Table-top micro-centrifuge for 1.5 ml tubes (e.g., Carl Roth Mini-Zentrifuge ROTILABO‱, cat. no. T464.1)
- Table-top micro-centrifuge for 5 ml tubes (e.g., myFuge 5D Digital 5mL Centrifuge, cat. no. C2595-E)
- Thermal PCR cycler (e.g., MJ Research, DNA Engine Thermal Cycler PTC-200, cat. no. 8252-30-0001)
- Plate sealer (e.g., Bio-Rad Px1 PCR plate sealer, cat. no. 1814000)
Bravo electronic protocol files
Appendix
Indexing primer sequences
8bp_iPCR_P5_P7_plateLayout_20211220SN.xlsx27KB Documents for buffers and reagents
Document
NAME
TE bufferCREATED BY
Anna Schmidt
Document
NAME
SPRI beads, variable PEG concentration
CREATED BY
Ancient DNA Core Unit
Documents for primer plates
Document
NAME
Bravo workstation: Preparation of indexing primer aliquots in 384-well plates
CREATED BY
Ancient DNA Core Unit
Indexing PCR setup
Indexing PCR setup
Before each run, follow the instructions in Labfolder and document the experiment.
Note
[Documentation]
Document the experiment in Labfolder.
[Note]
The Labfolder entry name consists of the name of the Labfolder template and the library plate ID. To document your experiment, fill the data element fields in Labfolder. They serve as a template for creating CoreDB entries later on. Providing the plate ID and performing the database work is usually done by the main person responsible for library preparation in the Core Unit, not by the person performing the experiment.
Thaw the following components at room temperature:
- Library plate(s) (up to four plates to be indexed with one set of indexing primers)
- 10x AccuPrime Pfx buffer
- P5 primer plate 384 well
- P7 primer plate 384 well
Note
[Note]
See MATERIALS for the preparation of the 384-well P5 and P7 primer plates. Do NOT vortex the plates after thawing.
Prepare PCR master mix in a 5 ml Eppendorf tube by combining the reagents below. Mix by flicking the tube with a finger and briefly centrifuge in a 5 ml table centrifuge.
| Reagent | Volume (μl) | Volume per reaction (μl) | Final concentration | |
| Water | 2200 | 20 | ||
| 10x AccuPrime Pfx buffer | 1100 | 10 | 1x | |
| AccuPrime Pfx DNA Polymerase | 110 | 1 |
Note
[Documentation]
Note down the lot/batch information of the reagents in the respective fields in Labfolder.
Pipette 422 µl of the master mix to each well of column 12 of a NUNC Plate. Pipette very slowly to prevent foaming. Leave the plate on the bench until used.
| 12 | |
| A-H | 422 µl |
Switch on all components of the Bravo system, including the external cooling device (set to 4 °C). Switch on light and ventilation ("Betrieb") inside the robot hood.
Log into the VWworks software using the administrator account (password "a") and open the protocol under "S:\Bravo_protocols\MPI-EVAN-homebrew\forms\ssDNA_Library_preparation\96_ssDNAprep_indexingPCR_8bp_indices.VWForm". Initialize the system.
Briefly centrifuge the library plate as well as P5 and P7 primer plates in a plate centrifuge to ensure that the foil is clean and free of liquid before you carefully remove the foil.
Set up the Bravo deck as indicated by the form file and specify the volume of library that will be indexed.
Note
[Note]
Default library input volume is 49 μl. If smaller volumes of library should be indexed, put an HTS deep well reservoir filled approximately half with water on position 1 of the Bravo deck. This reservoir will be used to substitute the reduced input volume.
Load a text file containing the desired combinations of indices for each reaction and associated Bravo pipetting instructions.
Note
[Note]
Input files are available at "P:\AncientDNA\indices\8bp_indices_correct", sorted alphabetically in the order in which they should be used. Pick the first file in the folder, rename it by adding the library plate ID (e.g., 01A_Pl_397.txt), and transfer it to the following folder: "P:\AncientDNA\indices\8bp_indices_correct\used_indices". From there, load it into the protocol.
CAUTION: In case the input file names contains "revP5" (e.g., "49A.revP5.txt"), the P5 primer plate needs to be rotated 180 ° on the deck, so that the numbers and letters are upside down.
Start the run by clicking the "start" button and follow the instructions by VWorks.
Note
[Note]
Run time is ca. 30 min. The first 15 min are used for aliquoting the P5 and P7 primers to the indexing PCR plate. After this, a pause point is reached, at which the user will be prompted to add the master mix if it has not been added already. Inexperienced users are advised to start the run only after all components have been added to the Bravo deck, as indicated in the form file.
Finishing indexing PCR setup and cycling
Finishing indexing PCR setup and cycling
After the run has finished, seal the PCR plate with peelable foil in the Bio-Rad plate sealer (185 °C, 3 s, use sealing frame). Store PCR plate in the fridge until it is transferred to the post-PCR lab for cycling. If less than 49 μl library were used for PCR, seal the library plate containing the leftover libraries (180 °C, 3 s, use sealing frame) and put it back to the freezer. If needed, go to step #1 to prepare additional indexing PCR plates using P5 and P7 indexing primers from the same set.
After all PCR plates have been prepared, discard all plastic ware that is still in the robot and clean the robot deck using ethanol and water. Switch off the robot and restart the computer.
Note
[Note]
If less than four indexing PCR plates were prepared, also discard the remaining indexing primer aliquots. Sealing and re-using primer aliquots is strongly discouraged due to the risk of cross-contamination between primers.
Take the indexing PCR plate(s) to a post-PCR lab equipped with thermo cyclers and start PCR cycling using the profile below.
Note
Cycling is performed using the following parameters:
| Step | Temperature °C | Time | |
| Initial denaturation | 95 | 2 min | |
| Per cycle denaturation | 95 | 20 s | |
| Per cycle annealing | 60 | 30 s | |
| Per cycle extension | 68 | 1 min | |
| Goto 2, 34 times | |||
| Final extension | 68 | 5 min | |
| Hold | 10 | forever |
- The cycling takes about 2 h 20 min.
Note
[Documentation]
Add cycler and room number to your entry in Labfolder.
When cycling has completed, store the PCR plate(s) in the fridge until indexing library purification is performed. Perform purification within one week.
SPRI purification set-up
SPRI purification set-up
Switch on all components of the Bravo system. The external cooling device is not needed for this protocol.
Log into the VWworks software using the administrator account (password "a") and open the automated purification protocol under "S:\Bravo_protocols\MPI-EVAN-homebrew\forms\SPRI_purification\V2_2_1_96_SPRI_33_Purification_Dilutions.VWForm". Initialize the system.
Take the indexing PCR plate out of the fridge and briefly centrifuge it in a plate centrifuge to make sure that there are no droplets remaining at the foil. Do not vortex the plate.
Specify the parameters in the menu of the form file.
Note
[Note]
The default values for post-indexing purification are as follows:
- Sample volume: 85 µl
- Elution volume: 40 µl
- Number of columns to be purified: 12
- Specify last filled column in tip box: 12
- Specify first filled column in tip box: 1
- Specify sample plate type (cannot be changed): Eppendorf twin. tec semi-skirted PCR plate
- Specify SPRI beads reservoir type: 96 Ay Deep-well HTS reservoir
Set up the Bravo deck as indicated by the form file.
Fill buffer reservoirs as listed in the table below and load them into the MiniHub as indicated by the form file.
| Reagent | Volume (ml) | |
| 80% EtOH | approx. 50 | |
| TE buffer | approx. 30 | |
| SPRI beads (33% PEG) | approx. 30 |
Note
[Note]
Mix the SPRI bead suspension thoroughly by shaking. Make sure that no bead pellet or clumps are remaining.
Note
[Documentation]
Note down the reagent batches in the respective fields in Labfolder.
Start the purification by clicking the "Run" button.
Note
[Note]
- Run time is approximately 1 h 20 min. The run starts with aliquoting 5 µl backup of the unpurified libraries into a fresh empty Eppendorf full skirted plate. This takes ca. 5 min. After this step the run will be paused automatically.
When prompted by the software, seal the plate with peelable foil in the Bio-Rad plate sealer (180 °C, 3 s, use sealing frame). Label the plate "5ul backup indexed & UNpurified" and store it in the freezer. Continue the run by clicking "Continue".
Finishing SPRI purification
Finishing SPRI purification
After the run has finished, seal the plates with peelable foil using the Bio-Rad sealer (180 °C, 3 s, use sealing frame).
Note
[Note]
There are three plates to store after library purification:
- The final indexed and purified library plate (40 µl). Label the plate with plate ID, "indexed and purified", the date and your initials. Store the plate in the fridge for subsequent generation of library pools. After pooling, the plate should be stored in the respective library box in the freezer.
- A 10-fold dilution plate (20 µl total volume), to be used as a template for reamplification if necessary. Label the plate with plate ID, "1:10 reamp backup", the date and your initials. Freeze it for later use.
- A 2.5-fold dilution plate (5 µl total volume), to be used for NanoDrop measurement. Label the plate with plate ID, "NanoDrop dilution indexed and purified', the date and your initials. Store the plate in the fridge until NanoDrop measurement is performed. Important: NanoDrop measurements should never be performed when the plate was already stored in the freezer!
Remove and discard all the plastic ware that is still in the robot. Discard any leftover reagents in liquid waste container and clean the robot using ethanol and water. Switch off the robot and restart the computer.
Indexing library quantification using Nanodrop device
Indexing library quantification using Nanodrop device
Take the 2.5-fold dilution plate for NanoDrop measurement from the fridge and briefly centrifuge.
Note
[Note]
To avoid freezing/thawing-induced concentration gradients, the indexed library plate should always be stored in the FRIDGE until the NanoDrop measurements are performed.
Start the NanoDrop Software on the computer linked to the NanoDrop device (ND 8000). Choose the 8-channel measurement for Nucleic Acid (DNA-50, dsDNA) and follow the instructions by the software.
Clean the NanoDrop device by adding 1 μl of water to the measurement pedestal using a multi-channel pipette. Close the pedestal and click "Ok" to initialize the instrument. Open it and clean it with a soft tissue.
Blank the device by adding 1 μl of TE buffer to the pedestal. Close the pedestal and click "Ok" to blank the device. Open the pedestal and clean again with soft tissue.
Note
[Documentation]
Note down the reagent information in the respective fields in Labfolder.
Carefully remove the plate seal of the NanoDrop dilution plate and measure the concentrations of the indexing libraries column-wise using the multi-channel pipette. Clean pedestal after every measurement with soft tissue.
Expected result
Purified indexed libraries typically yield DNA concentrations between 100 and 200 ng/μl (after correcting for the 2.5-fold dilution). Repeat measurements for samples with values below 50 ng/µl or samples showing unexpected curves.
Save the measurements as an MS Excel file in your personal folder on the public server (P:\user\Name). Save a copy on the Coreunit server in the respective library plate folder under "coreunit(V:)\SummaryTables\Library/Pl_XXX".
Note
[Note]
Attach the file to your entry in Labfolder.
Seal the 2.5-fold dilution plate with peelable foil (180 °C, 3 s, use sealing frame). This plate can be discarded after one year of storage. Label the plate with "Discard in MMYYYY" and put it into the corresponding drawer in the freezer.