Jul 27, 2023

Public workspaceRapid, high throughput library preparation of SARS-CoV2 using Illumina’s DNA Prep Library Preparation Kit V.3

DOI
https://dx.doi.org/10.17504/protocols.io.b3vgqn3w
  • Jason Nguyen1,
  • Rebecca Hickman1,
  • Natalie Prystajecky1,
  • John Tyson1,
  • Tracy Lee2
  • 1BCCDC Public Health Laboratory;
  • 2BCCDC
BCCDC Public Health Laboratory
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DOI: https://dx.doi.org/10.17504/protocols.io.b3vgqn3w
Protocol Citation: Jason Nguyen, Rebecca Hickman, Natalie Prystajecky, John Tyson, Tracy Lee 2023. Rapid, high throughput library preparation of SARS-CoV2 using Illumina’s DNA Prep Library Preparation Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.b3vgqn3wVersion created by MMG_Lab
Manuscript citation:
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: January 14, 2022
Last Modified: July 27, 2023
Protocol Integer ID: 56968
Keywords: SARS-CoV2, nCOV, COVID, COVID-19, whole genome sequencing, Illumina, illumina dna prep library preparation kit, dna prep library preparation kit this procedure, high throughput library preparation of sar, dna prep library preparation kit, dna library, dna libraries for whole genome, high throughput library preparation, genome sequencing, cov2, pcr protocol, sequencing, library preparation, post pcr, whole genome, sar, pooled post pcr
Abstract
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

Materials
ABC
Reagents Equipment Supplies
Illumina DNA Prep, (M) Tagmentation, 96 samples (Cat: 20018705) Post-PCR thermal cyclers Applied Biosystems Standard 96 well plates
Nextera UD Indexes A-D or custom IDT adapters Single and multichannel pipettes Adhesive plate seals and applicator
Nuclease-free water Vortex Reagent boats
Plate spinner Pipet tips; various sizes. Filter plugged and nuclease free
SPRI 96-well Magnetic Plate Disposable Powder-free Gloves and Gowns
Magnetic tube rack for 1.5ml tubes Discard pail and waste bags
1.5ml Lo-bind snap-top tubes
Troubleshooting
Tagment Genomic DNA
10s
Add Amount15 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.


Vortex the BLT and TB1 tubes vigorously for Duration00:00:10 to resuspend. Spin down the tubes, but only briefly to not sediment the beads.

10s
Combine the following volumes to prepare the tagmentation master mix: Reagent overage is included in the 96 sample volume to ensure accurate pipetting.


ABC
ReagentVolume per sampleVolume (96 samples)
Bead-Linked Transposome (BLT)5 µl565 µl
Tagmentation Buffer (TB1)5 µl565 µl
Final volume10 µl1130 µl

Vortex the tagmentation master mix thoroughly to resuspend. Divide the tagmentation master mix volume equally by pipetting Amount141.25 µL into each well of an 8-tube strip or alternatively add the master mix to a low dead volume reservoir.

Using a multichannel pipette, transfer Amount10 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.

Seal the plate with adhesive seal, spin down briefly, and run the TAG program on the thermal cycler as follows:


AB
TemperatureTime
55 C15 minutes
10 CHold

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

ABC
ReagentVolume per sampleVolume (96 samples)
Enhanced PCR Mix (EPM)10 µl1130 µl
Nuclease-free water10 µl1130 µl
Final volume20 µl2260 µl


Reagent overage is included in the volume to ensure accurate pipetting.
Vortex and centrifuge the PCR master mix.
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.

Remove the plate from the magnet.
Using a low dead volume reservoir and a multichannel pipette, immediately add Amount20 µL l of the PCR master mix directly onto the beads in each sample well. Pipette the mix until the beads are fully re-suspended.

Spin down your adapter plate before use. Using a multichannel pipette, puncture the foil seal of the adapter plate and add Amount5 µL of pre-pared i7 and i5 index adapters to each sample. Pipette 10 times to mix.

Seal the plate and spin down to remove as many bubbles as possible.
Place on the thermal cycler and run the BLT PCR program as follows:

ABC
TemperatureTimeCycles
68 C3 minutes1
98 C3 minutes1
98 C45 seconds8*
62C30 seconds
68C2 minutes
68 C1 minute1

*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.
Proceed to procedure "Clean Up Pooled Libraries"
Clean Up Pooled Libraries
18m 30s
Warm the Sample Purification Beads (SPB) to room temperature before use. Thaw the Resuspension Buffer (RSB) to room temperature before use.
Make Amount500 µL of 70-80% ethanol for washes.

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.
Put the plate on the magnetic stand until the liquid is clear (~Duration00:01:00 )

1m
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.
Add Amount40 µL of nuclease free water to a new 1.5mL Lo-bind tube labelled “1”.

Vortex and spin down the BLT Pool and then transferAmount45 µL to tube 1, the Lo-bind tube containing the water.

Vortex and invert the SPB tube to resuspend. Add Amount45 µL of SPB directly into tube “1” containing the BLT Pool and water. Mix by pipetting slowly 10 times, avoiding bubbles.

Incubate at room temperature for Duration00:05:00 .

5m
During this incubation, add Amount15 µL of SPB into a brand new 1.5mL tube labelled “2”

Once the 5 minute incubation is complete, place tube “1” on the magnet and wait until the liquid is clear (~1 minute).


Once the liquid is clear, transfer Amount125 µL of supernatant from tube “1” into tube “2” containing the Amount15 µL SPB. Pipette 10 times to mix, again slowly to avoid bubbles. Discard the original tube “1”.

Incubate tube “2” for another Duration00:05:00 .

5m
Place tube “2” on the magnetic stand until the liquid is clear (~1 minute).


Without disturbing the beads, remove and discard the supernatant out of tube “2”.
Perform two washes as follows:

With tube “2” remaining on the magnetic stand, add Amount200 µL of the previously made 70-80% ethanol to each well without mixing

Incubate for Duration00:00:30 Without disturbing the beads, remove and discard the supernatant. Repeat the wash a second time.

30s
Use a 20 µl pipette to remove and discard residual ethanol from each well.
Air-dry on the magnetic stand for Duration00:05:00 .

5m
Remove from the magnetic stand.
Using a single channel pipet, add Amount35 µL of RSB to the beads, pipetting up and down to resuspend, slowly.

Incubate at room temperature for Duration00:02:00 .

2m
Place tube “2” on the magnetic stand and wait until the liquid is clear (~2 minutes).


Transfer Amount30 µL of 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”.

If you are stopping, store your libraries in Temperature-20 °C freezer for up to a month.

If you are not stopping, proceed to the appropriate protocol for quantification, diluting, and denaturing as per your sample type.