TITLE: Bravo workstation: automated single-stranded DNA library preparation (ssDNA2.0)
AUTHORS: Sarah Nagel, Anna Schmidt, Ayinuer Aximu Petri, Anya Patova, Merlin Szymanski, Elena Essel, Matthias Meyer

[DESCRIPTION]
We here provide an implementation of the ssDNA2.0 single-stranded library preparation method (Gansauge et al. 2017, Gansauge et al. 2020) for automated liquid handling on the Bravo NGS workstation B in 96-well format. The method was developed primarily for ancient DNA research but is also suitable for library preparation from other sources of degraded or single-stranded DNA, such as cell-free DNA, formalin-fixed samples or synthetic oligonucleotides. 

To use the protocol, a Bravo NGS workstation is required. Calibration of the instrument for this protocol has to be performed by the user and requires significant expertise in using the platform. Electronic protocol files for the Bravo NGS workstation are provided together with this protocol and a brief description of the steps performed by the liquid handling system, potentially providing a framework for setting the method up on liquid handling systems from other manufacturers. 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. We strongly advise against manual execution of the protocol, as manual handling lacks the precision of automated liquid handling and may lead to cross-contamination of samples or non-optimal results. For manual ssDNA 2.0 library preparation, please refer to Gansauge et al. 2020. Note that the adapter sequences added with this method differ from standard Illumina adapters by a 5-bp deletion in the P5-adapter, requiring a custom sequencing primer (see Gansauge et al. 2020). 

This protocol consists of two parts: (i) library preparation, with or without Uracil-DNA-glycosylase treatment for the removal of internal uracils from ancient DNA molecules, and (ii) library quantification by quantitative real-time PCR. A protocol for library amplification and indexing is provided separately. 

References
Gansauge, M.-T., Gerber, T., Glocke, I., Korlevic, P., Lippik, L., Nagel, S., Riehl, L. M., Schmidt, A., &
Meyer, M. (2017). Single-stranded DNA library preparation from highly degraded DNA using T4 DNA
ligase. Nucleic Acids Research, 45(10): e79.

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.




[STEPS]
SECTION: Reagent and sample preparation (days/weeks/months before the run)
1. Prepare master mixes (Desphosphorylation mix, Adapter/splinter mix, Ligation mix I, Fill-in mix, Klenow mix, Ligation mix II) and aliquots of library positive controls (LPCs), library negative controls (LNCs) and if needed positive text extracts (PTEs) as described in MATERIALS. 
SECTION: Preparing and starting a library preparation run
11. Select the proper settings in the formfile:
FluidX tube type (by default select "96 Ay Brooks Fluidx 1ml tubes")
Spike-in (enable by clicking the box)
Type of buffer reservoir (usually HTS deep well reservoirs are used)
SECTION: Reagent and sample preparation (days/weeks/months before the run)
2. Decontaminate other reagents and consumables as described in MATERIALS. 
SECTION: Preparing and starting a library preparation run
9. Set up Bravo deck as indicated by the form file. The MiniHub can be filled later once the robot is already running.
SECTION: Preparing and starting a library preparation run
12. Start run by clicking the "Run" button and follow the instructions by VWorks. 
 

SECTION: Preparing and starting a library preparation run
14. Prepare 100 pM dilution of spike-in in a 50 ml Falcon tube by combining 20 ml of decontaminated TET-buffer and 2 μl of 1 μM oligonucleotide CL304. Mix well. Pour spike-in dilution into a buffer reservoir and load it into the MiniHub as indicated by the form file.

 

SECTION: Preparing and starting a library preparation run
13. Prepare streptavidin beads:
SECTION: Preparing and starting a library preparation run
13.1. Fully resuspend stock suspension of MyOne C1 beads by vortexing. Make sure no bead pellet remains.
SECTION: Preparing and starting a library preparation run
13.2. Transfer 1,200 μl stock suspension of beads into an Eppendorf 1.5 ml Safe-lock tube. Pellet beads in magnetic rack, pipette off and discard supernatant. Add an additional 1200 µl stock suspension of beads (2400 µl used in total), pellet the beads and discard the supernatant. 
SECTION: Preparing and starting a library preparation run
13.3. Wash beads twice with 1,000 µl 0.1xBWT buffer and discard the wash buffer. 
SECTION: Preparing and starting a library preparation run
13.4. Resuspend beads in 680 μl 0.1xBWT buffer and transfer the bead suspension to a 5 ml ThermoFisherScientific screw-cap tube. When prompted by VWorks (approximately 20 min after the run started), add the tube into the ThermoFisher Scientific 5 ml tube rack at the position indicated above.   

 

SECTION: Preparing and starting a library preparation run
15. Fill buffer reservoirs as listed in the table below and load them into the MiniHub as indicated by the form file.

 

 

SECTION: Preparing and starting a library preparation run
17. Add remaining consumables (PCR plates, waste plate) to the MiniHub and missing tip boxes into the stackers. Double-check that all required positions are correctly filled. 
SECTION: During the library preparation run
18. Observe parts of the run:
Always check if all master mixes were correctly pipetted to the NUNC master mix plate. To do so, pause the system when it doesn't carry tips or plastic ware by clicking "Pause". Check the NUNC plate, place it back and click "Continue". 
Check if all liquid was transferred from the FluidX tubes to the sample plate by watching this step (~75 min after the run started).
Approximately 30 min before the run ends, watch the elution step, especially the transfer of the eluate to the final library plate. Sometimes liquid may be left on the plate. This also becomes apparent by a lack of air in the tips, which is aspirated after the liquid. If the liquid transfer was incomplete, pause the robot once it finished the step and use a manual pipette to transfer remaining liquid to the library plate. Make sure to not aspirate beads. 
SECTION: Finishing the library preparation run
19. After the elution step, the run is automatically paused. Take out the final library plate from position 7 of the Bravo deck, seal it with peelable foil in the Bio-Rad plate sealer (180 °C, 3 s, use sealing frame) and briefly centrifuge it in a plate centrifuge to make sure there are no air bubbles at the bottom of the wells. Perform another visual check to make sure that all bubbles are gone. If necessary centrifuge again. Peel off the foil and place the plate back into position 7 of the Bravo deck. Press "continue". 
SECTION: Finishing the library preparation run
20. When the run is finished take out the final library plate (49 µl per well) and the 50x dilution plate (50 µl per well) and seal them with peelable foil (180 °C, 3 s, use sealing frame). Store the library plate and the 50x dilution plate at -20 °C in freezer 'Morpheus'. The 50x dilution plate will be transferred to the post-PCR lab for qPCR measurement later on. 
SECTION: Finishing the library preparation run
21. Discard all the plastic ware that is still in the robot. When doing so, check the plates for any remaining liquids to make sure that all pipetting steps were performed accurately. Collect liquid waste in 500 ml bottles next to the sink using a funnel. Discard used tips to regular trash and the tip boxes to the large waste bin on the left side of the cooling unit. 

 

SECTION: Finishing the library preparation run
22. Clean the robot by wiping the Bravo deck and the benches using tissues and ethanol. Switch off the robot and the cooling device and restart the computer so that remote access to the computer becomes possible. Switch off the computer screen.
SECTION: Library and spike-in quantification using qPCR
23. In the post-PCR lab, thaw the 50x library dilution plate and all required PCR reagents as well as the PCR strip tube containing the qPCR standard dilution (ranging from 102 to 108 copies/μl) at room temperature (qPCR reagents are stored in lab B4.12, freezer 26, drawer "qPCR/ddPCR stocks"). 

 

SECTION: Library and spike-in quantification using qPCR
24. Prepare qPCR master mixes for assay 1 (quantification of all molecules) and assay 2 (quantification of spike-in/library preparation efficiency) in 5 ml eppendorf tubes by combining the reagents below. Mix by vortexing and briefly centrifuge in a 5 ml table centrifuge.

 


 

SECTION: Library and spike-in quantification using qPCR
25. Pipette 320 µl of assay 1 master mix to each well of column 8 of a NUNC Plate. Pipette 264 µl of assay 2 master mix to each well of column 9. Keep NUNC plate in fridge until used.

 

SECTION: Library and spike-in quantification using qPCR
26. Switch on all components of the Bravo system, including the external cooling device (set to 4 °C).
SECTION: Library and spike-in quantification using qPCR
27. Log into the VWworks software using the administrator account (password "a"). Load the qPCR setup protocol under "S:\Bravo protocols\MPI-EVAN-homebrew\forms\qPCR_and_dilutions\V3_96_ssDNAprep_qPCR_setup.VWForm". Initialize the system.
SECTION: Library and spike-in quantification using qPCR
28. Select the proper settings in the form file:
Choose "96" from drop-down menu (there is also a legacy version of the protocol supporting qPCR measurements of half library plates).
SECTION: Library and spike-in quantification using qPCR
30. Set up Bravo deck as indicated by the form file.

SECTION: Library and spike-in quantification using qPCR
31. Start run by clicking the "Run" button and follow the instructions by VWorks.

 

SECTION: Library and spike-in quantification using qPCR
32. After the run has finished, take out the library dilution plate from position 7 of the Bravo deck, seal it with peelable foil in a Bio-Rad plate sealer (180 °C, 3 s, use sealing frame) and store it  at -20 °C in freezer 23, drawer "1:50 library dilutions", for future experiments. Take out the qPCR plate from position 5, seal it with optically clear foil (167 °C for 3 s, no sealing frame; make sure the glossy side faces up) and briefly centrifuge it to make sure there are no bubbles left in the wells.
SECTION: Library and spike-in quantification using qPCR
33. Transfer qPCR plate to the CFX384 Real-Time PCR system and perform the following steps using the BioRad CFX Manager software:
Go to "Run setup", choose "user defined"
from the "protocol" tab, choose "select existing" and select "CFX_3StepAmp+Melt_PROBE_10μl Assay.prcl" from "X:\public\AncientDNA\protocols\qPCR_Bravo\cycling program"
from the "plate" tab, choose "select existing" and select "96_Sample Plate 384wells_FAM.pltd" from "X:\public\AncientDNA\protocols\qPCR_Bravo\qPCR_platefile"
SECTION: Library and spike-in quantification using qPCR
34. Start the cycling and save the file in a new subfolder in your personal folder on the public server labeled with the respective library plate ID.

 

 

 

SECTION: Library and spike-in quantification using qPCR
35. Discard the qPCR plate and all the plastic ware that is still in the robot. Collect liquid waste in 500 ml bottles. Keep half filled tip box from stacker 2 (clean tips), cover tips and store tip box for later use.
SECTION: Reagent and sample preparation (days/weeks/months before the run)
3. Prepare a FluidX sample rack containing 96 sample DNA extracts and extraction negative controls in FluidX screw-cap tubes in 30 µl volumes. Store the rack at:
room temperature for same day library preparation
4 °C for next day library preparation
-20 °C for library preparation at a later time

[Option 1 ] DNA extraction and library preparation were planned together (default)

On the day of library preparation, add LNCs and LPCs to the existing FluidX sample rack as described below.    

[Option 2] Designing a new library preparation experiment

Follow the guidelines below. 

 

SECTION: Preparing and starting a library preparation run
6.3. Spin down master mix tubes in a table centrifuge for 5 ml tubes.  
SECTION: Preparing and starting a library preparation run
6.2. Mix the other master mixes by flicking the tubes with a finger. 
SECTION: Preparing and starting a library preparation run
6.4. Insert master mix tubes into the below-indicated positions of a ThermoFisher Scientific 5 ml tube rack. Before entering the tubes, make sure Microseal B Adhesive foil is attached to the bottom of the rack to secure the tubes in places during the run. Replace foil if it is no longer sticky. Store rack in the fridge until use.

 

 

SECTION: Preparing and starting a library preparation run
6.1. After adding T4 DNA ligase to Ligation mix I, rotate the tube for at least 10 min at room temperature in a tube rotator to ensure complete mixing of the highly viscous solution. 
SECTION: Preparing and starting a library preparation run
6. Complete master mixes by adding the following volumes of enzyme, starting with Ligation mix I. 
OPTIONAL: For UDG-treatment of ancient DNA, add USER enzyme mix to the Dephosphorylation mix.

 

 

SECTION: Preparing and starting a library preparation run
8. Log into the VWworks software using the administrator account (password "a"). Load the library preparation protocol under "S:\Bravo_protocols\MPI-EVAN-homebrew\forms\ssDNA_Library_preparation\96_ssDNAprep.VWForm". Initialize the system.
SECTION: Preparing and starting a library preparation run
7. 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. 
SECTION: Preparing and starting a library preparation run
10. Unpack 34 Bravo tip boxes. Remove the lids and load tip boxes into stackers 1-3 of the BenchCel as indicated in the form file. 
 

SECTION: Preparing and starting a library preparation run
5. Thaw the following components at room temperature:
master mixes (Desphosphorylation mix, Adapter/splinter mix, Ligation mix I, Fill-in mix, Klenow mix, Ligation mix II)
sample DNA extract rack
aliquot of a 1 μM spike-in oligonucleotide CL304 dilution
LNCs and LPCs as needed below   

 

SECTION: Preparing and starting a library preparation run
16. Prepare the sample DNA extract rack as follows:
SECTION: Preparing and starting a library preparation run
16.1. Start the FluidX barcoder reader software and allow the camera to initialize.
SECTION: Preparing and starting a library preparation run
16.3. Use the FluidX barcode reader to read the bottom barcodes of all tubes in the rack. 

 

SECTION: Preparing and starting a library preparation run
16.4. Vortex and spin down the sample rack. Open the FluidX screw-cap tubes in the sample rack with the decapper and discard the lids. Place the sample rack into the position in the MiniHub indicated by the form file.
SECTION: Preparing and starting a library preparation run
16.2. When working with a pre-configured library plate, add FluidX tubes with LNCs and LPCs to the sample rack by replacing tubes at designated 'space holder' positions using the information from the "samples_to_screen" list available at "P:\AncientDNA\samples". Store the 'space holder' tubes for use in future experiments. 

 

SECTION: Preparing and starting a library preparation run
4. Before each run, follow the instructions in Labfolder and document the experiment.

 

SECTION: Library and spike-in quantification using qPCR
29. Load stacker 3 with four full Bravo tip boxes (without lids) as indicated in the form file. Make sure that all the other stackers stay empty.
SECTION: Library and spike-in quantification using qPCR
36. Clean the robot by wiping the Bravo deck and the benches using tissues and ethanol. Switch off the robot and the cooling device and restart the computer so that remote access to the computer becomes possible. Switch off the computer screen.
SECTION: Library and spike-in quantification using qPCR
37. Perform data analysis on any computer connected to the network using the BioRad CFX Manager software.