TITLE: Miniaturized and Automatized Whole-Genome Amplification of SARS-CoV 2 Virus using Illumina CovidSeq reagents for Next-Generation Sequencing V.1
AUTHORS: Quentin Semanas, christophe GINEVRA, Richard Chalvignac

[DESCRIPTION]
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.



[STEPS]
SECTION: Abstract
1. 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. 


SECTION: Abstract
2. 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).
SECTION: Pre-PCR - Anneling - cDNA Synthesis - Amplification
4. Pre-PCR was prepared using Illumina COVIDSeq Assay (RUO) kit with a miniturized and automatized protocol using SPT Labtech automatons (Mosquito and Dragonfly).

 

 

SECTION: Pre-PCR - Anneling - cDNA Synthesis - Amplification
5. 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. 



 


 


 

SECTION: Pre-PCR - Anneling - cDNA Synthesis - Amplification
5.1. Annealing - Preparation

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

3.57 µL 
SECTION: Pre-PCR - Anneling - cDNA Synthesis - Amplification
5.2. RNA Extract Pool

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

3.57 µL  per Well
SECTION: Pre-PCR - Anneling - cDNA Synthesis - Amplification
5.3. Annealing 

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

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

 

SECTION: Pre-PCR - Anneling - cDNA Synthesis - Amplification
5.4. 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).

4.706 µL  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. 

5 µL  per Well
SECTION: Pre-PCR - Anneling - cDNA Synthesis - Amplification
5.5. 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 :

 

SECTION: Pre-PCR - Anneling - cDNA Synthesis - Amplification
5.6. 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.

14 µL  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. 

3.5 µL  per Well for the two amplification plates
SECTION: Pre-PCR - Anneling - cDNA Synthesis - Amplification
5.7. 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 :

 

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

 

 

SECTION: Post-PCR - Library Preparation
7. 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.



 


 

SECTION: Nucleic acids extraction
3. 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.
SECTION: Post-PCR - Library Preparation
7.1. 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 :

5 µL  into the Library Pool Plate

It will result of 10µL POOL 1/2 per well for 384 samples.
SECTION: Post-PCR - Library Preparation
7.2. Tagmentation 

Prepare two different new plates for mix distribution :

Eppendorf LoBind 384 Well Plates labelled "Tagmentation Plate"
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"

3 µL 

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

50 µL on green columns
40 µL on red columns

 
 
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".

2 µL into Tagmentation Plate

SECTION: Post-PCR - Library Preparation
7.3. Tagmentation incubation / Stop

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

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

 

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

1 µL  directly into the Tagmentation Plate

Seal, briefly vortex and centrifuge the plate. Incubate :

5 min 
SECTION: Post-PCR - Library Preparation
7.4. 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

2 min 

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

4 µL 

Add TWB in each well of the "Tagmentation Plate"

5 µL 

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

SECTION: Post-PCR - Library Preparation
7.5. Amplify and Index Tagmented Amplicons 

When washes steps are done :

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

4 µL into "Tagmentation Plate"
1 µL   into "Tagmentation Plate"

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


 

SECTION: Post-PCR - Library Preparation
7.6. 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" :

1 µL 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 :

192 µL 

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

172.8 µL  containing the 192µL Final Pool. 

Be careful to hardly vortex ITB before use.

Vortex to mix and incubate :

5 min 

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 :

55 µL 

Vortex and centrifuge briefly. Incubate :

2 min 

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.