Apr 18, 2026
Library preparation of SARS-CoV-2 RNA using Molecular Inversion Probes (MIPs) target capture for Whole Genome Sequencing (WGS) on Illumina platform
- 1The Wellcome – Wolfson Institute for Experimental Medicine, Queen’s University Belfast
Protocol Citation: Hortensia-Clara Radulescu, David Simpson 2026. Library preparation of SARS-CoV-2 RNA using Molecular Inversion Probes (MIPs) target capture for Whole Genome Sequencing (WGS) on Illumina platform. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqx18klk5/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: April 02, 2026
Last Modified: April 18, 2026
Protocol Integer ID: 314418
Keywords: SARS-CoV-2, Whole Genome Sequencing (WGS), Molecular Inversion Probes , MIPs, library preparation, Illumina sequencing, target capture, whole genome sequencing on an illumina miseq platform, target capture for whole genome sequencing, library preparation of sar, using molecular inversion probe, molecular inversion probe, whole genome sequencing, illumina miseq platform, sar
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Abstract
The protocol describes the steps for the library preparation of SARS-CoV-2 RNA using the Molecular Inversion Probes (MIPs) technology for target capture. The libraries can then be used in Whole Genome Sequencing on an Illumina MiSeq platform.
Guidelines
This protocol has been applied with all the different size designs of the MIPs of: 75nt, 150nt and 225nt available for Illumina platform.
Materials
Reagents
- Target Capture Kit Viral RNA, SC2-IL-75-1, ML5100-1062 (for 75nt probe design), Molecular Loop
- Target Capture Kit Viral RNA, SC2-IL-150-1, ML5100-1061 (for 150nt probe design), Molecular Loop
- TargetCapture Kit Viral RNA, SARS-CoV-2, ML5100-IL (for 225nt probe design), Molecular Loop
- Index Primer plate, 96 samples x1 rxn for Illumina, ML2100, Molecular Loop
- Kappa Hyper Pure beads (Roche)
- Qubit 1X dsDNA High Sensitivity (HS) (ThermoFisher Scientific)
- 80% ethanol
Consumables
- 96 well PCR plates
- sterile filter tips 10µl, 20µl, 200µl
- single channel pipettes 10µl, 20µl, 200µl
- 1.5 ml Eppendorf Lobind tubes
Equipments
- Minicentrifuge for Eppendorf tubes
- Vortex mixer
- Thermocycler for 96 well PCR plates
- Centrifuge for 96 well plates plates
- Agilent Tapestation 4200 System
- Qubit Fluorimeter (Invitrogen,ThermoFisher Scientific)
Protocol materials
Qubit™ dsDNA HS Assay KitInvitrogen - Thermo FisherCatalog #Q32851
D1000 Screen TapeAgilent Technologies
REVERSE TRANSCRIPTION (RT) and PROBE HYBRIDIZATION
SARS-CoV-2 RNA samples are taken out of the – -80 °C freezer and placed On ice .
The RT and probe hybridization reaction is set-up On ice in a 96 well PCR plate.
The following reagent tubes are taken out of the -20 °C freezer and put On ice to thaw: the RT Mix tube (transparent reagent) and Probe Mix viral RNA tube (blue reagent). The tubes are vortexed and spun down.
Prepare the RT-hybridization Master Mix in a 1.5ml Eppendorf tube, combining the following reagents volumes.
| A | B | C | D | E | |
| Component | 1x (µl) | 8x (µl) | 24x (µl) | 96x (µl) | |
| RT Mix | 1.6 | 12.8 | 38.4 | 153.6 | |
| Probe Mix | 0.4 | 3.2 | 9.6 | 38.4 | |
| Total | 2 | 16 | 48 | 192 |
Table 1. Reagents volumes for the RT-hybridization Master Mix
Note
For each Master Mix preparation from the protocol, depending on the number of samples and the appropriate reagent volumes needed, 20% of the final volume for each reagent should be added to count for the waste.
Transfer from the RT-hybridization Master Mix tube, for each sample, 2 µL into a well of the 96 PCR plate.
For each sample, add 6 µL RNA into a well of a 96 well PCR plate.
Mix by pipetting and shortly spin down the plate.
Note
All centrifugation steps for the reaction plate are performed at 280g x 30 sec unless otherwise defined.
Place the PCR plate in a thermocycler and incubate for 16 hours according to the cycling conditions defined in Table 2.
| A | B | |
| Temperature (°C) | Time (min) | |
| 25 °C | 10 min | |
| 50 °C | 50 min | |
| 95 °C | 1 min | |
| 55 °C | ∞ |
Table 2. Cycling parameters for the RT and hybridization reaction
Fill-IN REACTION
Remove from -20 °C freezer the Fill-in Mix Viral RNA tube (yellow reagent) and place it on On ice to thaw. Shortly vortex and spin down.
Note
It is important that the Fill-in Mix Viral RNA reagent is fully thawed and ready to be added to the reaction plate upon completion of the hybridization reaction.
Following the 16 hour incubation period, remove the reaction plate from the thermocycler, while keeping the hybridization program running at 55 °C
Spin down the reaction plate and remove the PCR seal.
Add 2 µL of the Fill-in reagent to each reaction on the plate. The color of the reaction is now pale green.
Cover the plate with a PCR seal, pulse vortex and shortly spin down.
Return the reaction plate to the thermocycler and allow the Fill-in reaction to proceed for 1 hour.
| A | B | |
| Temperature (0C) | Time (min) | |
| 55 °C | 1 h |
Table 3. Cycling parameters for the Fill-in reaction
CLEAN-UP and PCR REACTION
Remove from the freezer the Clean-up Mix Viral RNA tube (red colour) and the PCR Mix Viral RNA tube (transparent colour) and thaw on ice . After thawing, vortex and spin down. Remove from freezer the Illumina Index Primer plate and place it on ice to thaw. Pulse vortex and spin down.
Note
The Clean-up and PCR reaction must be set-up just at the time when the 1 hour Fill-in reaction has ended.
Prepare the Clean-up-PCR Master Mix mixing the volumes from Table 4 in a 1.5ml Eppendorf tube.
| A | B | C | D | E | |
| Component | 1x (µl) | 8x (µl) | 24x (µl) | 96x (µl) | |
| Clean-up Mix | 2 | 16 | 48 | 192 | |
| PCR Mix | 12 | 96 | 288 | 1152 | |
| Total | 14 | 112 | 336 | 1344 |
Table 4. Reagents volumes for the Clean-up and PCR Master Mix
Stop the Fill-in reaction and take the reaction plate out of the thermocycler. Spin down the reaction plate and remove the seal.
Add the following reagents to each reaction in the plate:
- 14 µL of the Clean-up-PCR Mix prepared before
- 2.4 µL of the appropriate index primer
Cover the plate with a PCR seal, pulse vortex and spin down.
Place the reaction plate back in the thermocycler and run a program with the cycling conditions from Table 5.
| A | B | C | |
| Cycles | Temperature (0C) | Time (min or sec) | |
| 1 | 45 °C | 15 min | |
| 1 | 95 °C | 3 min | |
| 25 | 98 °C | 15 sec | |
| 60 °C | 15 sec | ||
| 72 °C | 1 min | ||
| 1 | 4 °C | ∞ |
Table 5. Cycling parameters for the Clean-up and PCR reaction
After the ending of the Clean-up and PCR reaction program place the plate at -20 °C or proceed to the step 4 of the protocol.
LIBRARY POOLING and PURIFICATION
Spin down the reaction plate and remove the PCR seal.
Take the Kappa beads out of the fridge and leave them at least 30 min. at Room temperature
Pool an equal volume from each sample library into a 1.5ml Eppendorf tube, to obtain a total pool volume for purification of 100 µL .
Perform a 0.8x bead cleanup by adding 80 µL of Kappa beads to the pooled library and pipette mix thoroughly. Incubate 00:05:00 min at Room temperature .
Place the 1.5ml tube to a magnetic rack and incubate for 00:05:00 min at Room temperature .
Pipette off the supernatant and wash the pellet twice with 80% ethanol.
Allow the bead pellet to dry for approx 00:05:00 min atRoom temperature , ensuring not to overdry.
Elute in 20 µL Tris (10 mM, pH 8.0).
QC the final purified library before sequencing using Qubit™ dsDNA HS Assay KitInvitrogen - Thermo FisherCatalog #Q32851 on a Qubit Fluorimeter for the cleaned library concentration, and the D1000 Screen TapeAgilent Technologies on a Agilent Tapestation for finding the library fragment size.
Protocol references
Molecular Loop, Viral RNA Target Capture Kit. User Guide ML5000/ML5100/ML5200. Protocol version 2.6.0V