TITLE: Rapid, high throughput library preparation of SARS-CoV2 using Illumina’s DNA Prep Library Preparation Kit
AUTHORS: Jason Nguyen, Rebecca Hickman, Tracy Lee, Natalie Prystajecky, John Tyson

[DESCRIPTION]
This procedure provides instructions on how to prepare DNA libraries for whole genome sequencing on an Illumina MiSeq or NextSeq using Illumina’s DNA Prep Library Preparation Kit scaled to half reaction volumes with modifications to the post-PCR procedures; tagmentation stop buffer and associated washes are removed and libraries are pooled post PCR then a single size selection is performed.

This protocol is used to sequence SARS-CoV-2 using the cDNA/PCR protocol: https://dx.doi.org/10.17504/protocols.io.b3viqn4e


[STEPS]
SECTION: Tagment Genomic DNA
1. Add 15 µL of DNA to each well of 96 well PCR plate.  If the DNA volume is less than 15 µl, add nuclease-free water to the DNA samples to bring the total volume to 15 µl. 

Note: it is assumed that the samples from your PCR plate (labeled “ amplicon”) have at least 3.3ng/µl. 


SECTION: Tagment Genomic DNA
2. Vortex the BLT and TB1 tubes vigorously for 10 sto resuspend. Spin down the tubes, but only briefly to not sediment the beads.

SECTION: Tagment Genomic DNA
3. Combine the following volumes to prepare the tagmentation master mix: Reagent overage is included in the 96 sample volume to ensure accurate pipetting.


 

SECTION: Tagment Genomic DNA
4. Vortex the tagmentation master mix thoroughly to resuspend. Divide the tagmentation master mix volume equally by pipetting 141.25 µL into each well of an 8-tube strip or alternatively add the master mix to a low dead volume reservoir.

SECTION: Tagment Genomic DNA
5. Using a multichannel pipette, transfer 10 µL of the tagmentation master mix to each well of the plate, pipetting each sample 10 times to resuspend. Use fresh tips for each sample column.

SECTION: Tagment Genomic DNA
6. Seal the plate with adhesive seal, spin down briefly, and run the TAG program on the thermal cycler as follows:


 

SECTION: Amplify Tagmented DNA
7. Thaw the Enhanced PCR Mix (EPM) on ice, vortex, and briefly centrifuge. Thaw your plate of index adapters to room temperature.
SECTION: Amplify Tagmented DNA
8. Combine the following volumes to prepare the PCR master mix: 

 


Reagent overage is included in the volume to ensure accurate pipetting.
SECTION: Amplify Tagmented DNA
9. Vortex and centrifuge the PCR master mix.
SECTION: Amplify Tagmented DNA
10. Remove the BLT plate from the thermal cycler and place on a magnet. Once the liquid is clear, use a multichannel pipette to remove and discard the supernatant. 

SECTION: Amplify Tagmented DNA
11. Remove the plate from the magnet.
SECTION: Amplify Tagmented DNA
12. Using a low dead volume reservoir and a multichannel pipette, immediately add 20 µLl of the PCR master mix directly onto the beads in each sample well. Pipette the mix until the beads are fully re-suspended.

SECTION: Amplify Tagmented DNA
13. Spin down your adapter plate before use. Using a multichannel pipette, puncture the foil seal of the adapter plate and add 5 µLof pre-pared i7 and i5 index adapters to each sample. Pipette 10 times to mix.

SECTION: Amplify Tagmented DNA
14. Seal the plate and spin down to remove as many bubbles as possible.
SECTION: Amplify Tagmented DNA
15. Place on the thermal cycler and run the BLT PCR program as follows: 

 

*Depending on the total DNA input at the beginning of the experiment, the number of PCR cycles must be altered accordingly. For specimens >100ng, 5 PCR cycles must be performed. It was found with this rapid protocol that 8 PCR cycles produced the best results. After the BLT PCR program, libraries can be stored at 2-8C for up to 3 days.
SECTION: Amplify Tagmented DNA
16. Proceed to procedure "Clean Up Pooled Libraries"
SECTION: Clean Up Pooled Libraries
17. Warm the Sample Purification Beads (SPB) to room temperature before use. Thaw the Resuspension Buffer (RSB) to room temperature before use.
SECTION: Clean Up Pooled Libraries
18. Make 500 µL of 70-80% ethanol for washes.

SECTION: Clean Up Pooled Libraries
19. After the BLT PCR program has completed, remove the plate from the thermal cycler and quickly spin down to collect the contents to the bottom of the well.
SECTION: Clean Up Pooled Libraries
20. Put the plate on the magnetic stand until the liquid is clear (~1 min)

SECTION: Clean Up Pooled Libraries
21. Multi-channel 2 µl from each column of the BLT PCR plate into an 8-tube strip avoiding bead carry over, then combine all 8 of the tubes into a 1.5mL Lo-bind Eppendorf tube labelled with the run name followed by “BLT Pool” for long term storage. The BLT PCR plate can be discarded.
SECTION: Clean Up Pooled Libraries
22. Add 40 µLof nuclease free water to a new 1.5mL Lo-bind tube labelled “1”.

SECTION: Clean Up Pooled Libraries
23. Vortex and spin down the BLT Pool and then transfer45 µLto tube 1, the Lo-bind tube containing the water.

SECTION: Clean Up Pooled Libraries
24. Vortex and invert the SPB tube to resuspend. Add 45 µLof SPB directly into tube “1” containing the BLT Pool and water. Mix by pipetting slowly 10 times, avoiding bubbles.

SECTION: Clean Up Pooled Libraries
25. Incubate at room temperature for 5 min.

SECTION: Clean Up Pooled Libraries
26. During this incubation, add 15 µLof SPB into a brand new 1.5mL tube labelled “2”

SECTION: Clean Up Pooled Libraries
27. Once the 5 minute incubation is complete, place tube “1” on the magnet and wait until the liquid is clear (~1 minute).


SECTION: Clean Up Pooled Libraries
28. Once the liquid is clear, transfer 125 µLof supernatant from tube “1” into tube “2” containing the 15 µLSPB. Pipette 10 times to mix, again slowly to avoid bubbles. Discard the original tube “1”.

SECTION: Clean Up Pooled Libraries
29. Incubate tube “2” for another 5 min.

SECTION: Clean Up Pooled Libraries
30. Place tube “2” on the magnetic stand until the liquid is clear (~1 minute). 


SECTION: Clean Up Pooled Libraries
31. Without disturbing the beads, remove and discard the supernatant out of tube “2”.
SECTION: Clean Up Pooled Libraries
32. Perform two washes as follows: 

With tube “2” remaining on the magnetic stand, add 200 µL of the previously made 70-80% ethanol to each well without mixing 

Incubate for 30 s Without disturbing the beads, remove and discard the supernatant. Repeat the wash a second time.

SECTION: Clean Up Pooled Libraries
33. Use a 20 µl pipette to remove and discard residual ethanol from each well.
SECTION: Clean Up Pooled Libraries
34. Air-dry on the magnetic stand for 5 min.

SECTION: Clean Up Pooled Libraries
35. Remove from the magnetic stand.
SECTION: Clean Up Pooled Libraries
36. Using a single channel pipet, add 35 µL of RSB to the beads, pipetting up and down to resuspend, slowly.

SECTION: Clean Up Pooled Libraries
37. Incubate at room temperature for 2 min.

SECTION: Clean Up Pooled Libraries
38. Place tube “2” on the magnetic stand and wait until the liquid is clear (~2 minutes). 


SECTION: Clean Up Pooled Libraries
39. Transfer 30 µLof the supernatant to a new 1.5mL Lo-bind Eppendorf tube labelled with the Run ID and “Libraries” for long term storage. Dispose of tube “2”.

SECTION: Clean Up Pooled Libraries
40. If you are stopping, store your libraries in -20 °C freezer for up to a month.

SECTION: Clean Up Pooled Libraries
41. If you are not stopping, proceed to the appropriate protocol for quantification, diluting, and denaturing as per your sample type.