Jun 10, 2025

Public workspaceMiniaturized and Automatized Whole-Genome Amplification of SARS-CoV 2 Virus using Illumina CovidSeq reagents for Next-Generation Sequencing V.1 V.1

DOI
https://dx.doi.org/10.17504/protocols.io.14egn3zpzl5d/v1
Miniaturized and Automatized Whole-Genome Amplification of SARS-CoV 2 Virus using Illumina CovidSeq reagents for Next-Generation Sequencing V.1
  • Quentin Semanas1,2,
  • christophe GINEVRA1,2,
  • Richard Chalvignac1,2
  • 1Hospices Civils de Lyon;
  • 2genEPII
Quentin Semanas
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DOI: https://dx.doi.org/10.17504/protocols.io.14egn3zpzl5d/v1
Protocol Citation: Quentin Semanas, christophe GINEVRA, Richard Chalvignac 2025. Miniaturized and Automatized Whole-Genome Amplification of SARS-CoV 2 Virus using Illumina CovidSeq reagents for Next-Generation Sequencing V.1. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn3zpzl5d/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 19, 2023
Last Modified: June 10, 2025
Protocol Integer ID: 85227
Keywords: various next generation sequencing, viral genomic surveillance, rapid spread of severe acute respiratory syndrome coronavirus, severe acute respiratory syndrome coronavirus, genome amplification of sar, illumina covidseq reagents for next, sequencing strategy, using illumina covidseq reagent, sequencing protocol, miniaturization of sar, genome amplification, emergence of new variant, whole genome, cov, sar, emergence of variant
Abstract
Since December 2019, the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) around the world has necessitated to drastically scale-up the viral genomic surveillance to track the emergence of variant of concern. To scope with large-scale surveillance studies, various Next Generation Sequencing (NGS) methods have been reported and one of the main limitation has been the complexity of workflow in terms of turnaround time and cost effectiveness, limiting yield. By optimizing the Artic v4.1 sequencing strategy, we set-up automatization and miniaturization of SARS-CoV-2 whole genome sequencing protocol for monitoring emergence of new variants, applicable to a large workflow in a rapid and cost-effective manner.


Materials
ReagentIllumina CovidSeq AssayIllumina, Inc.
ReagentMGI Nucleic Acid Extraction KitMGI Tech CoCatalog #1000021043



Troubleshooting
Abstract
Since December 2019, the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) worlwide has necessitated to drastically scale-up the viral genomic surveillance to track the emergence of variant of concern.

The limitations of NGS workflow for increasing the genomic surveillance includes the cost effectivennes and the lack of automation of the technics used. By optimizing the Artic v4.1 sequencing strategy, we developped automatization and miniaturization of SARS-CoV-2 whole genome sequencing protocol for monitoring emergence of new variants, applicable to a large workflow in a rapid and cost-effective manner.

RNA extraction from nasopharyngeal swab was performed with the MGISP-960 workstation using MGI Easy Magnetic Beads Virus DNA/RNA Extraction Kit (MGI Tech, Marupe, Latvia).

The automatized and miniaturized method (Pre and Post PCR part) was performed with the Mosquito HV and Dragonfly‱ Discovery platforms (SPT Labtech, Melbourn, UK).
Each reagent distribution being performed with the Dragonfly and each pipetting step with the Mosquito HV excepted for the last Library Clean Up part of the process.


This protocol is suitable for miniaturized and automatized CovidSeq Illumina protocol on 384 samples (SARS-CoV 2). All reagents used are parts of Illumina COVIDSeq Assay (RUO) Kit excepted Amplification Primers (IDT ARTIC V4.1), UDI Indexes (IDT Indexes Illumina compatible).
Nucleic acids extraction
3h
Clinical SARS-CoV2 positive samples were extracted using MGI Nucleic Acid Extraction Kit (Catalog #1000021043) on MGISP 960 following manufacturer recommandations. It will provided a workflow of up to 192 samples in 1 hour and 20 minutes resulting in four 96 well RNA plates in 2 hours and 40 minutes.
3h
Pre-PCR - Anneling - cDNA Synthesis - Amplification
5h
Pre-PCR was prepared using Illumina COVIDSeq Assay (RUO) kit with a miniturized and automatized protocol using SPT Labtech automatons (Mosquito and Dragonfly).

Equipment
Mosquito HV
NAME
High Volume 16-Channel Robotic Liquid Handler
TYPE
SPT LabTech
BRAND
3097-01057
SKU
https://www.sptlabtech.com/products/liquid-handling/mosquito-hv/
LINK

Equipment
Dragonfly
NAME
Low volume liquid dispensing automaton
TYPE
SPT Labtech
BRAND
SU-0185
SKU
https://www.sptlabtech.com/products/dragonfly/dragonfly-discovery
LINK

PRE-PCR ASSAY

Prepare following mixes for each steps of Pre-PCR part (Annealing, RT, Amplification).
Volumes are edited for 384 samples including reagent overgage required by SPT automatons.



ReagentsVolume for 384 samples (µL)
EPH3 HT1500
Premix 1 - Annealing


ReagentsVolume for 384 samples (µL)
FSM HT1890
RVT HT210
TOTAL2100
Premix 2 - RT


ReagentsVolume for 384 samples (µL)
IPM HT3600
Primer Pool 1 or 2 (1/10)1032
NUCLEASE FREE WATER1128
TOTAL5760
Premix 3 - Amplification ( Do twice for each primer pool)

Annealing - Preparation

  • Using Dragonfly, distribute 3.57µL of EPH3 per well of an "Annealing" Eppendorf LoBind 384 Well Plate.

Amount3.57 µL EPH3
RNA Extract Pool

  • Using Mosquito HV 9mm, pool the four 96 Well RNA Plates into the "Annealing" Eppendorf LoBind 384 Well Plate.

Amount3.57 µL RNA per Well
Annealing

  • Seal, brielfly vortex the "Annealing" Eppendorf LoBind 384 Well Plate and centrifuge.

  • Place the plate in a thermocycler and run the following program :

TemperatureDuration
65°C5 min
4°CInfinite
Annealing Program - Indicate 7 µL as volume and heat lid at 75ºC.

RT-PCR - Preparation

  • When the thermal cycler program RT displays 5 min remaining, engage Premix 3 - RT distribution using the Dragonfly into a new Eppendorf LoBind 384 Well Plate ("RT" Eppendorf LoBind 384 Well Plate).

Amount4.706 µL RT Premix per Well

  • When the thermal cycler program Annealing is done, briefly centrifuge the plate.
Proceed to Mosquito HV 9mm transfer of Annealed RNA into the "RT" Eppendorf LoBind 384 Well Plate.

Amount5 µL Annealed RNA per Well
RT-PCR

  • Seal, brielfly vortex the "RT" Eppendorf LoBind 384 Well Plate and centrifuge.

  • Place the plate in a thermocycler and run the following program :

TemperatureDuration
25°C5 min
50°C10 min
80°C5 min
4°CInfinite
RT Program - Indicate 10 µL as volume and heat lid at 99ºC.

Amplification - Preparation

  • When the thermal cycler program RT displays 1 min remaining, engage Premix 2 - Amplification distribution for Primer Pool 1 and 2 using the Dragonfly into two new Eppendorf LoBind 384 Well Plate identifiate :

- "POOL 1" Eppendorf LoBind 384 Well Plate
- "POOL 2" Eppendorf LoBind 384 Well Plate.

Amount14 µL Amplification Premix per Well for Primer Pool 1 and 2

  • When the thermal cycler program RT is done, briefly centrifuge the plate.

  • Proceed to Mosquito HV 9mm transfer of cDNA into the two Amplification Eppendorf LoBind 384 Well Plate.

Amount3.5 µL cDNA per Well for the two amplification plates
Amplification

  • Seal, brielfly vortex the two Amplification Eppendorf LoBind 384 Well Plates and centrifuge.

  • Place the plate in a thermocycler and run the following program :

TemperatureDurationCycles
98°C3 min-
98°C15 sec32
63°C5 min
4°CInfinite -
Amplification Program - Indicate 17.5 µL as volume and heat lid at 100ºC.

Post-PCR - Library Preparation
3h
Sequencing library was prepared using Illumina COVIDSeq Assay (RUO) kit with a miniturized and automatized protocol using SPT Labtech automatons (Mosquito and Dragonfly).

Equipment
Mosquito HV
NAME
High Volume 16-Channel Robotic Liquid Handler
TYPE
SPT LabTech
BRAND
3097-01057
SKU
https://www.sptlabtech.com/products/liquid-handling/mosquito-hv/
LINK

Equipment
Dragonfly
NAME
Low volume liquid dispensing automaton
TYPE
SPT Labtech
BRAND
SU-0185
SKU
https://www.sptlabtech.com/products/dragonfly/dragonfly-discovery
LINK

POST-PCR ASSAY

  • Prepare following mixes for each steps of Post-PCR part (Tagmentation, Indexing PCR).
Volumes are edited for 384 samples including reagent overgage required by SPT automatons.

  • Washes and Tagmentation Stop steps reagents will be distributed directly into an new LVSD 384 well Plate using Dragonfly.



ReagentsVolume for 384 samples (µL)
ELBTs160.4
TB1481.3
NUCLEASE FREE WATER802.1
TOTAL1443.8
Premix 1 - Tagmentation


ReagentsVolume for 384 samples (µL)
EPM1200
NUCLEASE FREE WATER1200
TOTAL2400
Premix 2 - Amplification / Indexing PCR

Amplification POOL 1 and POOL 2 Eppendorf LoBind 384 Well Plates Pool

  • Following Amplification program, brielfly centrifuge the two Amplification POOL 1 and POOL 2 Eppendorf LoBind 384 Well Plates and place them on Mosquito HV 4.5mm board in order to proceed to Pool step :

  • Identify a new Eppendorf LoBind 384 Well Plates as " Library Pool Plate" and place it on Moquito HV 4.5mm board with the two Amplification Eppendorf LoBind 384 Well Plates.

  • Proceed to transfer :

Amount5 µL from each Pool plates (POOL 1 and 2) into the Library Pool Plate

  • It will result of 10µL POOL 1/2 per well for 384 samples.
Tagmentation

  • Prepare two different new plates for mix distribution :

  1. Eppendorf LoBind 384 Well Plates labelled "Tagmentation Plate"
  2. LVSD 384 well Plate labelled "Reagent / Trash Plate" (Half of this plate will be used for distribution of TWB and ST2 reagents; the other half will be used as trash for washes steps)

  • Using Dragonfly, proceed to :

1- Premix 1 distribution on "Tagmentation Plate"

Amount3 µL of ELBTs per well

2- Premix 2 distribution on "Reagent / Trash Plate" organized as follow :

Amount50 µL per well for TWB on green columns
Amount40 µL per well for EPM on red columns

"Reagent / Trash Plate" Organization
When distribution is done, place Plates on Mosquito HV 4.5mm board and proceed to transfer of Amplicons from "Library Pool Plate" to the "Tagmentation Plate".

Amount2 µL of SARS-CoV2 amplicons into Tagmentation Plate

Tagmentation incubation / Stop

  • When Mosquito tranfer is done, directly proceed to Tagmentation step.

  • Place the plate in a thermocycler and run the following program :

TemperatureDuration
55°C5 min
10°CInfinite
Tagmentation Program - Indicate 5 µL as volume and heat lid at 80ºC - Proceed to thermal cycler pre heat befrore to launch.

  • When Tagmentation is done, directly proceed to Stop step using Dragonfly :

Amount1 µL of ST2 per well directly into the Tagmentation Plate

  • Seal, briefly vortex and centrifuge the plate. Incubate :

Duration00:05:00 Room Temperature
5m
Critical
Post Tagmentation Clean Up

  • When Tagmentation is stopped, proceed to washes steps using Mosquito :

  • Place "Tagmentation Plate" on Mosquito 384 Magnetic Block
  • Incubate 384 Magnetic Block

Duration00:02:00 384 Magnetic Block

  • Remove supernatant and transfer it to "Reagent / Trash Plate"

Amount4 µL removed from "Tagmentation Plate" and transfer to "Reagent / Trash Plate"

  • Add TWB in each well of the "Tagmentation Plate"

Amount5 µL of TWB into "Tagmentation Plate"

  • Seal the plate, vortex and brilefly centrifuge.
  • Proceed to Wash Step once again until last supernatant removing

2m
Amplify and Index Tagmented Amplicons

When washes steps are done :

  • Removed supernatant
  • Proceed to Mosquito transfer of Premix 2 (EPM) AND indexes :

Amount4 µL of Premix 2 into "Tagmentation Plate"
Amount1 µL of Indexes into "Tagmentation Plate"

  • Seal, briefly vortex and centrifuge the plate.
  • Place the plate in a thermocycler and run the following program :


TemperatureDurationCycles
72°C3 min-
98°C 3 min-
98°C20 sec 7
60°C30 sec
72°C1 min
72°C3 min-
10°C Infinite-
Amplification / Indexing Program - Indicate 7 µL as volume and heat lid at 100ºC.

Library Pool and Manual Clean Up

  • When Amplification/Indexing Program is done, proceed to Pool of the 384 wells of "Tagmentation Plate" into a new Eppendorf LoBind 384 Well Plate labelled "Library Pool Plate" :

Amount1 µL of the 384 wells into one column of the new "Library Pool Plate"

  • Pool 12 µL of the 16 wells of the "Library Pool Plate" column into one Eppendorf LoBind 1.5mL Tube. It will result of a final volume :

Amount192 µL Pool Library

  • Proceed to Manual Clean Up using Illumina Tune Beads (ITB) at 0.9x. Add :

Amount172.8 µL of ITB into the Eppendorf LoBind 1.5mL containing the 192µL Final Pool.

Be careful to hardly vortex ITB before use.

  • Vortex to mix and incubate :

Duration00:05:00 at Room Temperature

  • Centrifugre briefly and place the Tube on magnetic stand until liquid is clear.
  • Remove supernatant and proceed to two EtOH 80% washes steps (30 sec contact, remove supernatant X2)
  • Let beads dry 1min after last superntaant removing
  • Add RSB to proceed to Elution :

Amount55 µL of RSB

  • Vortex and centrifuge briefly. Incubate :

Duration00:02:00 at Room Temperature

  • Place the Tube on magnetic stand until liquid is clear.
  • Transfer 50µL of supernatant into a new Eppendorf LoBind 1.5mL labelled "Final Library Pool"

Store on ice or at +4°C until Quantification Process following Illumina COVIDSeq Assay (RUO) Kit recommandations.

7m
Protocol references