Oct 18, 2021

Public workspaceUW Virology Swift SNAPv2 protocol

This protocol is a draft, published without a DOI.
  • Lasata Shrestha1,
  • Hong Xie1,
  • Shah A. Mohamed Bakhash1,
  • Robert J. Livingston1,
  • Meei-Li Huang1,
  • Alexander L. Greninger1,
  • Pavitra Roychoudhury1
  • 1Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA
Lasata Shrestha
Icon indicating open access to content
QR code linking to this content
Protocol Citation: Lasata Shrestha, Hong Xie, Shah A. Mohamed Bakhash, Robert J. Livingston, Meei-Li Huang, Alexander L. Greninger, Pavitra Roychoudhury 2021. UW Virology Swift SNAPv2 protocol. protocols.io https://protocols.io/view/uw-virology-swift-snapv2-protocol-byw4pxgw
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: October 10, 2021
Last Modified: October 18, 2021
Protocol Integer ID: 53948
Keywords: whole genome sequencing, SARS-CoV-2, SWIFT Biosciences, PerkinElmer, automation, genome
Abstract
Viral whole genome sequencing (WGS) has been instrumental in outbreak investigations, deployment of public health interventions, development as well as evaluation of vaccines and therapeutics. While multiple methods are commercially available for WGS, multiplex amplicon method has proven to be faster, more efficient, scalable, and more cost-effective compared to other methods. Here, we describe the automation of a multiplex amplicon panel for WGS of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), the causative agent of the current COVID-19 pandemic. The SWIFT Biosciences' primer set amplifies 345 amplicons designed against the SARS-CoV-2 Wuhan-Hu-1 complete genome (NC_045512.2), in a single tube to cover the ~30 kb SARS-CoV-2 genome in less than three hours.
Materials

Before start
The critical steps are tagged. Steps 5-108 are optional (for troubleshooting).
Single-strand cDNA synthesis Reagent Set-up
Single-strand cDNA synthesis Reagent Set-up
Prepare reagents for required number of samples as per the manufacturer's instructions (including 10% overage) linked below.
https://assets.thermofisher.com/TFS-Assets/LSG/manuals/SSIV_Reverse_Transcriptase_UG.pdf

Pipetting
Single-strand cDNA (sscDNA) synthesis Reagent Sciclone Set-up
Single-strand cDNA (sscDNA) synthesis Reagent Sciclone Set-up
Select the correct protocol on the liquid handler (Perkin Elmer Sciclone) and load the Sciclone as instructed.

This program will perform steps corresponding to steps 3 to 20 in the manual protocol. The code can be obtained from PerkinElmer.
Note
The SuperScript RT IV protocol was automated and optimized by Perkin Elmer personnel following ThermoFisher's commercially available protocol linked below.
SuperScript RT IV


Fig. 01: Empty Sciclone deck.
Equipment
Sciclone G3 NGSx iQ Workstation
NAME
Automated Liquid Handling
TYPE
Perkin Elmer
BRAND
CLS145321
SKU
https://www.perkinelmer.com/category/automation-liquid-handling-instruments
LINK
Fig. 02 Final (fully set-up) Sciclone desck set-up for sscDNA synthesis.


Critical
Sciclone movement: sscDNA synthesis
Sciclone movement: sscDNA synthesis
Moved Plate from D4 to B4 (magnet with no spacer)
Optional
Moved Plate from D2 to D4
Aspirate Amount11 µL of RNA from B4 and Dispense to plate on D4

Pipette mix and shake for Duration00:01:00

1m
Plate on B4 moved to C0 concurrently
Plate on C0 discarded
Moved plate from D4 to onboard thermocycler

Move ODTC lid from A1 to thermocycler
Thermocycler door closes and runs Duration00:07:40

7m 40s
Thermocycler door opens
ODTC lid moved from Thermocycler to A1
Moved plate from Thermocycler to D4
Empty tip box discarded. Fresh tip box placed onto C3
Load new tips
Aspirate Amount7 µL of PCR master mix from A4 and dispense into plate on D4

Pipette mix and shake for Duration00:01:00

1m
Move plate from D4 to onboard thermocycler
Move ODTC lid from A1 to thermocycler
Thermocycler door closes and runs for Duration00:53:04

53m 4s
Thermocycler door opens
Plate moved from thermocycler to D4
End product of Amount20 µL

Swift SNAP v2 Reagent Set-up
Swift SNAP v2 Reagent Set-up
Prepare reagents for required number of samples as per the manufacturer's instructions (including 10% overage) linked below.
Swift Normalase™ Amplicon Panels (SNAP) SARS-CoV-2, Additional Genome Coverage, and SARS-CoV-2 S Gene Panel
Pipetting
Swift SNAP v2 Sciclone Set-up
Swift SNAP v2 Sciclone Set-up
Select the correct protocol on the liquid handler (Perkin Elmer Sciclone) and load the Sciclone as instructed.

The selected program will perform steps corresponding to steps 23 to 127. The code can be obtained from PerkinElmer.


Note
The SWIFT SNAP v2 protocol was automated and optimized by Perkin Elmer personnel following SWIFT Bioscience's commercially available protocol linked below.
SWIFT SNAP v2


*Insert labeled photo of the deck without components (deck only)*
Fig. 03. Empty Sciclone deck.

*Insert labeled photo of the deck with labeled components and volume information*
Fig. 04. Final (fully-loaded) deck set-up for SWIFT SNAP v2 protocol on the Sciclone.
Equipment
Sciclone G3 NGSx iQ Workstation
NAME
Automated Library Preparation
TYPE
Perkin Elmer
BRAND
********
SKU
https://www.perkinelmer.com/category/automation-liquid-handling-instruments
LINK


Critical
Sciclone Movements: Swift SNAP v2 Multiplex PCR
Sciclone Movements: Swift SNAP v2 Multiplex PCR
1h 20m
1h 20m

Note
Steps 23-127 include detailed Sciclone deck movements for troubleshooting.

Optional
Moved Lid from A4 to A2
Pipette multiplex master-mix ( A4 to samples on D4)
Dispose off tips
Lid from A2 to A4
Mix samples on D4
Tips disposed
Shake mix of D4 (Noted: 30uL)
D4 to Thermocycler
ODTC lid placed
Thermo-cycler cover closedDuration01:15:00

1h 15m
PCR door opened
ODTC lid removed
The PCR plate was moved to D4
Moved plate from B2 to A3
Mix beads in B2, 10 X
Picked up 30 uL Beads from B2 to D4Amount30 µL Beads

Mixed D4 wells, 10 X
Shake mix D4 for several minutes
Plate moved from D4 to B4
Allow beads to sediment Duration00:05:00

5m
Supernatant discarded
Tips discarded
Alcohol cover B5 removed to C5
Fresh tips picked.
Picked up 80%ETOHAmount150 µL 80% Ethanol

Dispensed to D2
Supernatant removed to Wash plate
Tips disposed
Fresh tips picked
Alcohol (150uL) dispensed to plate B4Amount150 µL

Removed supernatant from B4
Tips discarded
Fresh tips picked
Alcohol transferred to B4Amount150 µL 80% EtOH

Supernatant collected and disposed
Tips discarded
Alcohol lid replaced back
Sample plate moved to D4
New tips
TE buffer lid
TE buffer addedAmount17.4 µL NEED TO CHECK

Mixed
Tips discarded
Plate D4 shake mixed
Lid from A4 lifted
Transfer master mix A4 to A3Amount28.9 µL Indexing PCR Reaction Mix

Tips disposed
Sciclone Movements: SWIFT SNAP v2 Indexing PCR
Sciclone Movements: SWIFT SNAP v2 Indexing PCR
1h 20m
1h 20m
Pipette Index from D2 to A3  ( 3.7 uL of indexes)
Optional
Pipette master mix plus indexes  from A3 to D4
Shake mixed D4
Lid of A2 to A4
Fresh tips
D4 mixed by pipetting
Tips disposal
D4 shake mixed
D4 plate moved to thermocycler
21 min PCR runDuration00:21:00

21m
Incubation 26 minDuration00:26:00

26m
Thermocycler door open
OPTC lid removed
Sample plate moved to D4
Beads plate moved from B2 to D3
Pipetted PEG NaCl from plate on B2 to plate on D4Amount32.5 µL PEG NaCl (ratio: 0.65)

Mixing in D4 by pipetting (10 times)
Shake mixed D4, for 4 min 35 secDuration00:04:35

4m 35s
Plate moved from D4 to C4 (on the magnet)
Incubation at room temperature Duration00:05:00

5m
Supernatant  from C4 discarded
Tips discarded
Alcohol lid removed
New tips picked
Picked up alcoholAmount150 µL 80% EtOH

Transferred alcohol to C4
1 min wait
Supernatant discarded
Tips  dropped off
New tips picked up
Picked up alcoholAmount150 µL 80% EtOH

Dispensed to C4
1 min wait
Supernatant discarded
Tips discarded
Moved Tip container from C3  to D0
New tip container picked up from A0
New tips picked up
Alcohol picked upAmount150 µL 80% EtOH

Dispensed alcohol to C4
1 min wait
Supernatant from C4  pipetted up and discarded
Tips discarded
Alcohol cover replaced
2 min wait (incubate at room temperature until residual alcohol evaporates)
Moved Plate  from C4 to D4
Picked up TE
Transfer TE to D4 Amount20 µL TE

Mixing
Tips discarded
Mix by pipetting and shaking in plate D4
Moved plate D4 to B4
Moved empty Plate A2 to D4
Tips picked up
Transfer from B4 to D4 Amount20 µL Eluate

Tips discarded
Plate lid from A4 to D4
Library Quality Control I
Library Quality Control I

Following the manufacturer's instructions for library quality control, quantify (Qubit or other fluorometric instruments) and determine the size (TapeStation or other electrophoretic instruments) of the library.
Equipment
4200 TapeStation System
NAME
Electrophoresis tool for DNA and RNA sample quality control.
TYPE
TapeStation Instruments
BRAND
G2991AA
SKU
https://www.agilent.com/en/product/automated-electrophoresis/tapestation-systems/tapestation-instruments/4200-tapestation-system-228263
LINK


Equipment
Invitrogen™ Qubit™ 3 Fluorometer
NAME
Accurately measures DNA, RNA, and protein using the highly sensitive fluorescence-based Qubit quantitation assays
TYPE
Invitrogen™ Q33216
BRAND
Q33216
SKU
https://www.fishersci.co.uk/shop/products/qubit-3-0-fluorometer/15387293
LINK



Critical
Normalase
Normalase
Following manufacturer's instructions as linked below, normalize the libraries using SWIFT's proprietary enzymatic normalization.
Normalase Pages: 9-12
Critical
Pooled Library Quality Control II
Pooled Library Quality Control II
Determine the library concentration for final library quality control before loading on the sequencer.
Quality Control Page: 12
Equipment
Invitrogen™ Qubit™ 3 Fluorometer
NAME
Accurately measures DNA, RNA, and protein using the highly sensitive fluorescence-based Qubit quantitation assays
TYPE
Invitrogen™ Q33216
BRAND
Q33216
SKU
https://www.fishersci.co.uk/shop/products/qubit-3-0-fluorometer/15387293
LINK

Critical
Sequencing
Sequencing
Following manufacturer's recommendation, load the pooled library for sequencing on the selected/preferred sequencer.
Sequencing Page: 13
Equipment
NextSeq 500 System
NAME
Sequencer
TYPE
Illumina
BRAND
*********
SKU
https://www.illumina.com/systems/sequencing-platforms/nextseq.html
LINK

Critical