Created by Commercial Training and Enablement 200003651 v01

Illumina® COVIDSeq™
Assay (96 samples) –
RUO Workflow

Presenter Name | Job Title

Date

For Research Use Only. Not for use in diagnostic procedures.

© 2021 Illumina, Inc. All rights reserved.

 Created by Commercial Training and Enablement
200003651 v01
Session Objectives

• By the end of this training, you will be able to:

• Define the Illumina COVIDSeq – RUO Kit Options
• Understand the Illumina COVIDSeq Assay – RUO workflow
• List best practices for processing samples, RNA extraction, cDNA synthesis, and
library preparation
• Understand the requirements of sample preparation prior to library preparation
• Describe the library preparation protocol steps
• Describe quantification and pooling steps for Illumina COVIDSeq Assay – RUO Kit

For Research Use Only. Not for use in diagnostic procedures.

 Created by Commercial Training and Enablement
200003651 v01

Illumina COVIDSeq RUO Kit Options

Illumina COVIDSeq Assay-- RUO Illumina COVIDSeq Test – RUO

96 sample size kit 3072 sample size kit
Reagents are contained in 4 boxes Reagents are in 4 boxes and labeled with HT
Positive Control sold separately Positive Control included
Index plate included Index Plates sold separately

• Low Throughput (LT) Sequencing • High Throughput (HT) Sequencing

Up to 28-48 samples per run Up to 3072 samples per run Up to 384 samples per run
• MiSeq System • NovaSeq 6000 System • NextSeq 500/550 System
• MiniSeq System • NextSeq 550Dx (RUO mode)
• iSeq 100 System • NextSeq2000 System

For Research Use Only. Not for use in diagnostic procedures.

 Created by Commercial Training and Enablement
200003651 v01

Flexibility with Illumina COVIDSeq RUO Kits

RNA extraction from decontaminated nasopharyngeal (NP), oropharyngeal (OP), and nasal swab
samples, as well as mid-turbinate specimens collected from individuals who meet COVID-19 clinical or
epidemiological criteria, using extraction kits such as the Quick-DNA/RNA Viral Magbead Kit

COVIDSeq Perform Surveillance

Assay –
RUO or
COVIDSeq Local DRAGEN COVID Pipeline with the COVID
Test-RUO Analysis Options:
Lineage Tools
Kit Cloud-Based DRAGEN COVID Lineage app on  Recommended for
BaseSpace Sequence Hub COVIDSeq Assay

Qualitative Detections
COVIDSeq
Test-RUO
of SARS-CoV-2 RNA
Kit Local DRAGEN COVIDSeq Test Pipeline
Analysis Options:
Cloud-Based DRAGEN COVIDSeq Test app on
BaseSpace Sequence Hub

For Research Use Only. Not for use in diagnostic procedures. 4

 Created by Commercial Training and Enablement

Illumina COVIDSeq – RUO Kit 200003651 v01

Workflow Overview
Library
Extract and cDNA Library
Sample Collection Amplify cDNA Quantification
Anneal RNA synthesis Preparation
and Pooling

pre amplification (amp) pre amp post amp

SAFE STOPPING POINT

Process
Samples

COVIDSEQ COVIDSEQ COVIDSEQ COVIDSEQ Tag &

Anneal Program FSS Program PCR Program Tag PCR Programs
SAFE STOPPING POINT SAFE STOPPING POINT SAFE STOPPING POINT
 Created by Commercial Training and Enablement
200003651 v01

Guidelines for Optimal Assay Performance

For optimal assay performance,

reference the following sections:
• Input Recommendations
AVOID CONFIRM KIT REAGENT STORAGE • Library Prep Introduction
CONTAMINATION CONTENTS AND AND HANDLING
REQUIRED CONSIDERATIONS • Tips and Techniques
CONSUMABLES

FOLLOW THE USE A FOLLOW SAFE

PROTOCOL AS UNIDIRECTIONAL STOPPING POINTS
WRITTEN LAB FLOW
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200003651 v01

Tips and Techniques

• Sealing and Unsealing the Plate • Centrifugation

• Seal plate before shake, vortex, centrifuge, and • Centrifuge as needed to consolidate liquid or beads
thermal cycle steps in the bottom of the well, and to prevent sample loss
• Apply seal to plate wells and around edges with a • Handling Beads
rubber roller
• Pipette slowly and mix thoroughly
• Centrifuge prior to unsealing plates
• Confirm no beads remain in the pipette tips after
• Unseal plate on a flat surface resuspension and mixing steps
• Plate Transfers • Washing beads steps:
• Confirm volume and well from each plate you are • Magnet best practices- use appropriate magnet,
removing and transferring samples keep on magnet as specified for each step
• If beads are aspirated into the pipette tips, dispense • Plate best practices- dispense liquid so all sides of
wells are wet, do not agitate plate while on magnet
back to the plate on the magnetic stand and wait stand, do not disturb the bead pellet
until the liquid is clear (~2 minutes)
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200003651 v01

Illumina COVIDSeq Assay – RUO for Surveillance

Quality Control Recommendations

Use of quality controls to evaluate assay performance is recommended

96 sample plate example Recommended Quality Controls

No Template Control (NTC)

94 patient samples - ELB provided
NTC
Positive Control- optional
positive control
+ CPC purchased separately

24, 48, or 96 sample plates: Note: Illumina recommends using one or

Only 22, 46, or 94 patient samples will be tested when including 1 NTC
more No Template Control to monitor
and 1 positive control
contamination

For Research Use Only. Not for use in diagnostic procedures.

 200003651 v01

Specimen
Collection
 Created by Commercial Training and Enablement

Illumina COVIDSeq RUO Kit 200003651 v01

Sample Collection
Library
Extract and cDNA Library
Sample Collection Amplify cDNA Quantification
Anneal RNA synthesis Preparation
and Pooling

pre amp pre amp post amp

Process
Samples

COVIDSEQ COVIDSEQ COVIDSEQ COVIDSEQ Tag &

Anneal Program FSS Program PCR Program Tag PCR Programs
 Created by Commercial Training and Enablement
200003651 v01

Sample Collection, Transportation, and Storage

Collection Transport Storage

CovidSeq Assay supports Transport samples according to Store samples according to the
patient samples derived from applicable governing instructions from the
nasopharyngeal (NP), regulations manufacturer of tubes used for
oropharyngeal (OP), and nasal sample transport
swabs
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Process Samples

Exceeding the storage times can negatively impact test results

Sample factors affecting SARS-CoV-2 detection

Sample collection methods, patient factors,

and/or the stage of the infection

Viral RNA degradation during shipping and

storage. RNA degradation can produce false-
negative results
• Handle all specimens as infectious agents
 200003651 v01

Extract and Anneal

RNA
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Illumina COVIDSeq RUO Kit 200003651 v01

Extract and Anneal RNA

Library
Extract and cDNA Library
Sample Collection Amplify cDNA Quantification
Anneal RNA synthesis Preparation
and Pooling

pre amp pre amp post amp

Process
Samples

COVIDSEQ COVIDSEQ COVIDSEQ COVIDSEQ Tag &

Anneal Program FSS Program PCR Program Tag PCR Programs
 Created by Commercial Training and Enablement

COVIDSeq Assay - RUO Quality Control Preparation 200003651 v01

Preparation Click image to play video of CPC Dilution

Positive Control - optional*

• Follow appropriate dilution of
COVIDSeq Positive Control (CPC)
with Elution Buffer (ELB )
No Template Control
• ELB used as no template control

If you plan to use the Procedure Best Practices

COVIDSeq Positive Control,
Positive Control* Handling CPC
make sure to follow the
• Extract RNA- • Do not allow multiple freeze-thaw
appropriate preparation
Follow 3 step dilution of CPC with ELB cycles
procedure given the workflow • Aliquot into low-bind tubes
step • Anneal RNA-
Follow 2 step dilution of CPC with ELB • Store at -85°C to -75°C
• Vortex before each use
* Prepare CPC prior to starting protocol steps

For Research Use Only. Not for use in diagnostic procedures.

 Created by Commercial Training and Enablement

Extract RNA 200003651 v01

Preparation Best Practices

Consumables Use No Template Control to detect
reagent contamination
• [Quick-DNA/RNA Viral MagBead]
2000 µl 96-well plate
• [QIAamp Viral RNA Mini Kit] 1.7 ml
Recommendation to include at least
LoBind Tubes
one NTC and one positive control.

Procedure
This step extracts RNA from Use COVIDSeq Extraction protocol specification within Illumina COVIDSeq RUO Kits
decontaminated viral transport Reference Guide
medium tubes • Follow Zymo Quick-DNA/RNA Viral MagBead for extraction steps
For each sample, add 400 μl patient sample to a new deep-well plate. If you plan to use controls, include
one tube of dilution 3 CPC (positive control) and ELB (no template control) per sample batch

• Follow Qiagen QIAamp Viral RNA Mini Kit for extraction steps
For each sample, add 140 μl patient sample to a 1.7 ml microcentrifuge tubeIf you plan to use controls,
include one tube of dilution 3 CPC (positive control) and ELB (no template control) per sample batch
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Quick-DNA/RNA Viral MagBead

Purify viral RNA using the MagBead protocol
• The sample is first lysed under highly denaturing conditions to inactivate RNases and to ensure
isolation of intact viral RNA
• In a deep-well plate, combine 400 μl patient sample and 800µl Viral DNA/RNA Buffer and mix well
Lysis (include patient samples, NTC, and optional CPC as positive control)

• Fully resuspend MagBinding beads prior to use*

• Add 20 μl MagBinding beads to each well, mix well by pipetting up and down ten times
Bind • Shake plate at 1500 rpm for 10 minutes

• Contaminants are efficiently washed away in two steps using two different wash buffers
Wash

• High quality RNA eluted in a special RNase-free water, ready for direct use or safe storage**
Elution
*Note: Important to fully resuspend MagBinding beads throughout Binding steps **Follow Quick-DNA/RNA Viral MagBead product documentation regarding
SAFE STOPPING POINT
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200003651 v01

QIAamp Viral RNA Mini Kit

Purify viral RNA using the spin protocol
• Sample lysed under highly denaturing conditions to inactivate RNases and isolate intact viral
RNA
Lysis • Include patient samples, NTC, and optional CPC as positive control

• Buffering conditions adjusted to provide optimum binding of RNA to QIAamp membrane

• Sample loaded onto the QIAamp Mini spin column
Bind • RNA binds to the membrane

• Contaminants are efficiently washed away in two steps using two different wash buffers
Wash

• High quality RNA eluted in a special RNase-free buffer, ready for direct use or safe storage*
• Purified RNA is free of protein, nucleases and other contaminants
Elution • Incubate elution for at least 1 minute and Elute in 30 µl Buffer AVE instead of 60 µl
*Follow QIAamp Viral RNA Mini Kit documentation regarding SAFE STOPPING POINT
 Created by Commercial Training and Enablement

Anneal RNA 200003651 v01

Preparation • Thaw EHP3 at room

temperature
Reagents
• Invert to mix EPH3
• Elution Primer
Fragment 3HC • Label new PCR plate
Mix (EPH3) CDNA1
• Confirm COVIDSEQ
Anneal thermal cycler
program

Click to play video of workflow step

Procedure
During this process, the extracted • Option to add CPC as positive control
RNA is annealed using random at this step
hexamers to prepare for cDNA • Add EPH3, eluted samples into CDNA1
synthesis
• Completely seal, shake and centrifuge
plate
• Run COVIDSEQ Anneal thermal cycler
program- preheat lid
 200003651 v01

Synthesize First
Strand cDNA
 Created by Commercial Training and Enablement

Illumina COVIDSeq RUO Workflow 200003651 v01

cDNA Synthesis
Library
Extract and cDNA Library
Sample Collection Amplify cDNA Quantification
Anneal RNA synthesis Preparation
and Pooling

pre amp pre amp post amp

Process
Samples

COVIDSEQ COVIDSEQ COVIDSEQ COVIDSEQ Tag &

Anneal Program FSS Program PCR Program Tag PCR Programs
 Created by Commercial Training and Enablement

Synthesize First Strand cDNA 200003651 v01

Preparation • Prepare Enzymes (FSM and RVT)

Reagents • FSM- thaw at room temperature,
invert to mix, keep on ice
• First Strand Mix (FSM)
• Reverse Transcriptase (RVT)
• RVT- invert to mix, keep on ice
• Prepare First Strand cDNA Master Mix
(MM)
• Confirm COVIDSEQ FSS thermal cycler
program

This step reverse transcribes the Procedure Click to play video of workflow step

RNA fragments primed with • Add First Strand MM to

random hexamers into first strand CDNA1 plate
cDNA using reverse transcriptase • Completely seal, shake and
centrifuge plate
• Run COVIDSEQ FSS thermal
cycler program- preheat lid
SAFE STOPPING PONT
If you are stopping, seal the plate and store at -25°C
to -15°C for up to 7 days
 200003651 v01

Amplify cDNA
 Created by Commercial Training and Enablement

Illumina COVIDSeq RUO Workflow 200003651 v01

Amplify cDNA
Library
Extract and cDNA Library
Sample Collection Amplify cDNA Quantification
Anneal RNA synthesis Preparation
and Pooling

pre amp pre amp post amp

Process
Samples

COVIDSEQ COVIDSEQ COVIDSEQ COVIDSEQ Tag &

Anneal Program FSS Program PCR Program Tag PCR Programs
 Created by Commercial Training and Enablement

Amplify cDNA 200003651 v01

• Prepare Reagents
Preparation • CPP1 and CPP2: thaw at room
Reagents temperature, keep on ice
• IPM: thaw at room temperature, invert
• COVIDSeq Primer Pool 1 (CPP1) to mix, keep on ice
• COVIDSeq Primer Pool 2 (CPP2) • Carefully label two new PCR plates
• Illumina PCR Mix (IPM) COV1 and COV2*
• Nuclease-free water • Confirm COVIDSeq PCR thermal cycler
program

Procedure Click to play video of workflow step.

• Prepare COVIDSeq PCR 1 Master Mix
This step uses two separate and COVIDSeq PCR 2 Master Mix –
PCR reactions to amplify Mix thoroughly
cDNA • Prepare COV1 and COV2 plates*
• Pre-PCR area: Properly seal plates
to avoid evaporation, shake and
centrifuge
• Post-PCR area: Run COVIDSEQ PCR
SAFE STOPPING POINT thermal cycler program- preheat lid
If you are stopping, seal the plate and store at
-25°C to -15°C for up to 3 days * The plates represent two separate PCR reactions on each sample and control in the CNDA1 plate
 200003651 v01

Illumina
Tagmentation
Library Preparation
 Created by Commercial Training and Enablement
200003651 v01

Illumina COVIDSeq RUO Workflow

Library Preparation
Library
Extract and cDNA Library
Sample Collection Amplify cDNA Quantification
Anneal RNA synthesis Preparation
and Pooling

pre amp pre amp post amp

Process
Samples

COVIDSEQ COVIDSEQ COVIDSEQ COVIDSEQ Tag &

Anneal Program FSS Program PCR Program Tag PCR Programs
 Created by Commercial Training and Enablement
200003651 v01

Library Prep is Critical for Successful Sequencing

For clustering: For sequencing: For mixing

Libraries must have P5 Libraries must have samples:
and P7 binding regions sequencing primer Libraries must have a
on either end of a library binding regions unique index or
barcode sequence
 Created by Commercial Training and Enablement
200003651 v01

Illumina Tagmentation Library Prep

Illumina Tagmentation Library Preparation Workflow

01 Tagment PCR Amplicons

PCR Amplicons

02 Post Tagmentation Clean up

03 Amplify and Add Adapters

04 Pool and Clean up Libraries

 Created by Commercial Training and Enablement
200003651 v01

Tagment PCR Amplicons

Preparation
• Prepare Reagents: Bring to room temperature, vortex to mix
• Prepare Tagmentation Master Mix (MM)
• Carefully label TAG1 plate
PCR Amplicons • Confirm COVIDSeq TAG thermal cycler program
Reagents
This step uses enrichment BLT to tagment PCR • Enrichment BLT (EBLTS)
amplicons, which is a process that fragments and tags • Tagmentation Buffer 1 (TB1)
the PCR amplicons with adapter sequences. • Nuclease-free water

Click to play video of the workflow step. Procedure

• Combine COV1 and COV2 plates into TAG1 plate
• Incubate Tagmentation MM + PCR amplicon product
• 50µl reaction of amplicon product and Tagmentation
MM (PCR plate)
• Completely seal and thoroughly shake plate
• Run COVIDSeq TAG thermal cycler program- preheat lid
• 55⁰C, 5 min → 10⁰C hold
 Created by Commercial Training and Enablement
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Tagment PCR Amplicons Best Practices

Best Practices
Handling eBLT Thermal cycle programming

• Bring to room temperature • Carefully label both plates

• Vortex eBLT before use for complete resuspension • Ensure TAG program at 10⁰C before
• Store above 2⁰C and upright to keep beads removing samples from thermal cycler
submerged in buffer
• Pipette slowly and mix thoroughly
• Confirm no beads remain in the pipette tips after
resuspension and mixing steps
• If beads are adhered to the side or top of the 96-well
plate, centrifuge at 500 × g for 1 minute, and then
pipette to resuspend

For Research Use Only. Not for use in diagnostic procedures.

 Created by Commercial Training and Enablement
200003651 v01

Post Tagmentation Clean Up

Preparation
Prepare Reagents: Reagents
• ST2: Vortex before use, confirm • Stop Tagmentation Buffer (ST2)
no presence of precipitates • Tagmentation Wash Buffer (TWB)
present
• TWB: Vortex before use
This step washes the adapter-tagged
amplicons before PCR amplification

Procedure Click to play video of the workflow step.

• Add ST2 to reaction, seal and shake
• Incubate plate at room temperature
for 5 minutes
• Using TWB wash beads two times
• To prevent over drying pellet, leave
TWB in plate until next step

For Research Use Only. Not for use in diagnostic procedures.

Confidential. Do not distribute. 32
 Created by Commercial Training and Enablement
200003651 v01

Post Tagmentation Clean Up Best Practices

Best Practices Prepare Reagents:

• ST2: Vortex before use,
Click to play video of TWB Best Practices. confirm no presence of
precipitates present
• TWB: Vortex • Avoid sample
before use loss- dispense ST2 and
This step washes the adapter-tagged TWB slowly to minimize foaming
amplicons before PCR amplification • Dispense TWB directly onto the beads
• Avoid bubbles
• Do not allow pellet to over dry

For Research Use Only. Not for use in diagnostic procedures.

Confidential. Do not distribute. 33
 Created by Commercial Training and Enablement
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Amplify Tagmented Amplicons

Preparation
• Prepare reagents- Keep EMP on ice Reagents
• Prepare PCR Master Mix (EPM and
Nuclease-free water) • Enhanced PCR Mix (EPM)
• Index adapters (IDT for Illumina PCR
Indexes Set 1, 2, 3, 4)
• Nuclease-free water

This step amplifies the

tagmented amplicons using a
PCR program. Procedure Click to play video of workflow step.
The PCR step adds paired • With plate on magnet remove all TWB
10 base pair Index 1 (i7) • Add PCR MM to TAG1 plate
• Add Indexes to TAG1 plate
adapters, Index 2 (i5) • Use one index set for each 96-
adapters, and sequences well sample plate
required for sequencing • Run COVIDSeq TAG PCR thermal
cluster generation. cycler program- preheated lid
• Confirm program is set with
72°C for 3 minutes incubation
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200003651 v01

Amplify Tagmented Amplicons Best Practices

Best Practices
Click to play video of Index plate Best Practice.

• Make sure all TWB has been removed from

samples before adding PCR master mix
• Invert EPM to mix
• Confirm resuspension after shaker mixing, if not
fully mixed, pipet to resuspend beads
• Index adapter plates
• Do not add samples to index plate wells
• Index plate wells cannot be reused
• Puncture foil seal prior to use
• Avoid index cross contamination
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200003651 v01

IDT for Illumina PCR Indexes Sets 1-4

10 base pair indexes
Ensure sample multiplexing for reliable downstream analysis

Unique Dual Indexes (UDI)

• Provided in 96-well plates
• Each well is single use only
• Ensure sample sheet includes 10
cycles of sequencing per index read

Each well contains unique combination of indexes

i.e. UDI pair (i5 + i7) pre-mixed in each well (example shown here)

For Research Use Only. Not for use in diagnostic procedures.

 Created by Commercial Training and Enablement
200003651 v01

Pool and Clean Up Libraries

Prepare Illumina Tune Beads (ITB) Tube
Preparation
• Prepare Illumina Tube Beads (ITB)
• Prepare pooled libraries in tube labelled Pooled
ITB Tube according to the appropriate sequencing
Library Plate Pooled ITB Tube system
This step combines libraries from each 96-well sample
plate into one 1.7 ml tube

Click to play video of the workflow step.

Procedure
• Combine individual sample reactions into ITB tube
• Transfer 5 μl library from each well of the PCR plate into
tube labelled Pooled ITB tube
• The final volume result depends on number of samples pooled
• MiSeq v2 flow cell- pool 15 samples
• MiSeq v3 flow cell- pool 24 samples
• MiniSeq HO flow cell- pool 24 samples
• iSeq 100 flow cell- pool 4 samples
 Created by Commercial Training and Enablement
200003651 v01

Pooling Guidance based on Sequencing System

For each sequencing system, the following table shows the recommended number of
samples required per flow cell based on 2x151 sequencing read length

Sequencing System Volume of Normalized Libraries (µl) Samples per Flow Cell (FC)
COVIDSeq Assay

MiSeq v2 Flow Cell 5 15

MiSeq v3 Flow Cell 5 24
MiniSeq HO Flow Cell 25 24

ISeq 100 Flow Cell 7.5 4

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Pool and Clean Up Libraries 200003651 v01

Bind optimal libraries to beads Preparation

• Prepare Reagents:
• ITB: Vortex to fully mix
• RSB: Bring to room temperature for 30 minutes, vortex
and invert to mix
• Prepare 2.5 ml 80% EtOH from absolute EtOH for each tube of
Pooled Add Vortex, Place on Remove
spin, and magnet until and discard pooled libraries
ITB ITB
supernatant
Tube incubate clear Reagents
Libraries of optimal size are then bound to magnetic • Illumina Tune Beads (ITB)
beads and fragments too small or large are wash away • Resuspension Buffer (RSB)

Click to play video of the workflow step. Procedure

• Perform clean up on pooled library tube
• Add ITB using the resulting volume of Pooled ITB tube
volume multiplied by 0.9
• Example Volumes:
• For 15 samples- add 67.5 µl ITB into each tube
• For 24 samples- add 108 µl ITB into each tube
 Created by Commercial Training and Enablement
200003651 v01

Pool and Clean Up Libraries

Wash away non optimal fragments
CAPTURE OPTIMAL Procedure:
FRAGMENTS
• Perform wash steps on magnet with freshly
• Add beads
made 80% EtOH
• Mix well
Bind • Incubate
• Elute library pool using RSB and transfer 50µl to
a new tube
• Pellet on magnetic stand
Click to play video of the workflow step.
ON THE MAGNET
• Remove supernatant
Wash • Wash with fresh 80% ethanol
• Do not disturb pellet
• Remove ethanol and air dry
CLEAN UP FINAL LIBRARY
• Add RSB
Elute • Mix well
• Incubate
• Pellet on magnet
• Elute sample from beads Note: Due to library yield excess, the RSB volume does not
impact batches with a small number of samples
 Created by Commercial Training and Enablement
200003651 v01
Pool and Clean Up Libraries Best Practices
Best Practices
Follow protocol as written ITB Handling Ethanol Washes

• Follow times and • Bring beads to room • The ethanol wash should be
temperatures for procedure temperature done with freshly made 80%
(as they may vary from other • Store ITB at 4°C ethanol from absolute ethanol
protocols) • Vortex for complete • Work quickly to prevent over
• Be cautious of tracking resuspension prior to use drying the pellet
sample during transfer step • Pipet slowly due to the
• Confirm sequencing system solution being viscous
and recommended number of
samples per flow cell

For Research Use Only. Not for use in diagnostic procedures.

 200003651 v01

Library
Quantification
and Pooling
 Created by Commercial Training and Enablement

Illumina COVIDSeq RUO Workflow 200003651 v01

Library Quantification and Pooling

Library
Extract and cDNA Library
Sample Collection Amplify cDNA Quantification
Anneal RNA synthesis Preparation
and Pooling

pre amp pre amp post amp

Process
Samples

COVIDSEQ COVIDSEQ COVIDSEQ COVIDSEQ Tag &

Anneal Program FSS Program PCR Program Tag PCR Programs
 Created by Commercial Training and Enablement
200003651 v01

Quantify and Normalize Libraries

Quantification Normalization
Quantification steps should be performed for Calculate the molarity using the formula provided
each pooled library tube

• Reagents Required:
• Qubit® dsDNA HS Assay Kit
• Qubit Assay Tubes
• Use 400bp as the average library size
• Equipment Required: • Dilute each library pool to a minimum of 30 µl at a
• Qubit Fluorometer 3.0 normalized concentration 4 nM using RSB

• Follow Qubit protocol as written

• If libraries are outside the standard range, dilute to
1:10 concentration, and analyze again
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200003651 v01

Pool and Dilute Libraries

Pool Libraries

This step pools and dilutes libraries to the starting concentration for sequencing system

Normalized Library Pool Dilute Libraries

• Transfer the designated volume of normalized libraries • Follow the denature and dilute instructions for system to
containing appropriate index adapter sets (i.e. set 1, 2, 3, dilute starting 4nM concentrations to the final loading
concentration
4) in a new microcentrifuge tube
• The final loading concentration is dependent on the
• Do not combine pools with the same index adapter sequencing system
set • MiSeq v2 flow cell- Final loading concentrations 10 pM
• This step produces a final pool of samples diluted to • MiSeq v3 flow cell- Final loading concentrations 12 pM
a starting concentration of 4nM • MiniSeq HO flow cell- Final loading concentrations 1.2 pM
• iSeq 100 flow cell- Final loading concentrations 75 pM
Sequence libraries as soon as possible after pooling
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Follow Loading Concentrations for Sequencing System

Dilute Libraries

Follow the denature and dilute instructions for system to dilute starting 4nM
concentrations to the final loading concentration

Sequencing System Volume of Normalized Libraries (µl) Final Loading Concentration (pM)
COVIDSeq Assay

MiSeq v2 Flow Cell 5 10

MiSeq v3 Flow Cell 5 12
MiniSeq HO Flow Cell 25 1.2
iSeq 100 Flow Cell 1.87 75

Final loading concentrations are a starting point and general guidelines. Optimize concentrations for your workflow over subsequent sequencing runs.

● Adjustments to final loading concentration should follow the denature and dilute instructions for your sequencing system

For Research Use Only. Not for use in diagnostic procedures.

 Created by Commercial Training and Enablement
200003651 v01

Sample Sheet Requirements

Use appropriate samplesheet.csv file for your sequencing system to create a sample sheet

Use this file as a template to create a

sample sheet

• https://support.illumina.com/downloads
/illumina-covidseq-test-sample-
sheet.html

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200003651 v01

Preparing for Sequencing on MiSeq System

Prepare Library Pool Sequencing Consumables Set up Sequencing Run

• Pool, Normalize to 4nM, and Dilute Set up Run in Manual Mode (unless
• MiSeq Reagent Kit v2 (300-cycle) performing surveillance and using
• MiSeq v2 flow cell- 15 samples Local Run Manager)*
• MiSeq Reagent Kit v3 (600-cycle) For Illumina DRAGEN COVIDSeq Test
• MiSeq v3 flow cell- 24 samples
BaseSpace Sequence Hub app
• Reference MiSeq System Guide
• Final loading concentration of • Select Run Monitoring and Storage as the
(document # 15046563) Configuration Option
pooled libraries
• Select Paired End as Read Type
• MiSeq v2 flow cell- 10 pM
• Run set up:
• MiSeq v3 flow cell- 12 pM • **Refer to the Illumina Technical • Read 1 and 2 – Enter appropriate
Note Sequencing Guidelines for read length**
• Reference MiSeq System Denature COVID-19 Surveillance Using the • Index 1 and Index 2 – Enter 10
and Dilute Libraries Guide (document Illumina COVIDSeq Test (RUO • *Refer to the Local Run Manager
# 15039740) Version) for guidance on read Software Guide (document #
length 1000000111492)

48
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200003651 v01

Preparing for Sequencing on MiniSeq System

Prepare Library Pool Sequencing Consumables Set up Sequencing Run

Set up Run in Manual Mode (unless
• Pool, Normalize to 4nM, and Dilute • MiniSeq High Output Reagent Kit performing surveillance and using
• MiniSeq HO flow cell- 24 samples (300-cycle) Local Run Manager)*
For Illumina DRAGEN COVIDSeq Test
• Final Loading Concentration of • Reference MiSeq System Guide BaseSpace Sequence Hub app
COVIDSeq Assay pooled libraries (document # 15046563)
• Select Run Monitoring and Storage as the
Configuration Option
• MiniSeq HO flow cell- 1.2 pM
• Select Paired End as Read Type
• **Refer to the Illumina Technical • Run set up:
• Reference MiniSeq System Note Sequencing Guidelines for • Read 1 and 2 – Enter appropriate
COVID-19 Surveillance Using the read length**
Denature and Dilute Libraries
Guide (document # Illumina COVIDSeq Test (RUO • Index 1 and Index 2 – Enter 10
1000000002697) Version) for guidance on read • *Refer to the Local Run Manager
length Software Guide (document #
1000000111492)

49
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Preparing for Sequencing on iSeq100 System

Prepare Library Pool Sequencing Consumables Set up Sequencing Run

Set up Run in Manual Mode (unless
• Pool, Normalize to 4nM, and Dilute performing surveillance and using
• iSeq100 i1 Reagent v2 Kit
Local Run Manager)*
• iSeq100 flow cell- 4 samples
• Reference iSeq100 Sequencing For Illumina DRAGEN COVIDSeq Test
• Final Loading Concentration of System Guide (document # BaseSpace Sequence Hub app
COVIDSeq Assay pooled libraries 1000000036024) • Select or enable Run Analysis,
Collaboration, and Storage in the System
• ISeq 100 flow cell- 75 pM Settings
• Select Paired End as Read Type
• **Refer to the Illumina Technical • Run set up:
• Reference iSeq100 Sequencing Note Sequencing Guidelines for • Read 1 and 2 – Enter appropriate
System Guide (document # COVID-19 Surveillance Using the read length**
1000000036024) Illumina COVIDSeq Test (RUO • Index 1 and Index 2 – Enter 10
Version) for guidance on read
length • *Refer to the Local Run Manager
Software Guide (document #
1000000111492)

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Illumina COVIDSeq RUO Analysis Options

After sequencing completes, analysis either takes place locally using installed pipeline software
or in BaseSpace Sequence Hub
• NovaSeq and NextSeq 1000/2000 sequencing systems
• Cloud-based analysis in BaseSpace Sequence Hub can use the Illumina DRAGEN COVIDSeq Test for
qualitative detection of SARS-CoV-2 or the DRAGEN COVID Lineage app for surveillance
• Local analysis for qualitative detection of SARS-CoV-2 RNA uses Illumina DRAGEN COVIDSeq Test
Pipeline
• All sequencing systems
• Local analysis for surveillance uses the Illumina DRAGEN COVID Pipeline with COVID Lineage Tools

• Refer to one of the following resources for more information

• Illumina DRAGEN COVIDSeq Test Pipeline Software Guide (document # 1000000128122)
• Illumina DRAGEN COVIDSeq Test App Guide (document # 1000000129548)
• Illumina DRAGEN COVID Pipeline Software Guide (document # 1000000158680)

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Illumina Application Technical Notes

• Safeguard experimental
results with proper
reagent handling
• Understand the impact
of genome coverage on • Maximize productivity with
SARS-CoV-2 variant aliquot volumes that
analysis match experimental
workflows
Sequencing guidelines for COVID-19
surveillance using the Illumina • Preserve reagent
COVIDSeqTM Test (RUO Version) effectiveness with best-
Technical Note
practice tracking and
storage procedures
Aliquot procedure for Illumina
COVIDSeq™ Test (RUO Version) kit
reagents Technical Note
For Research Use Only. Not for use in diagnostic
procedures.
 Created by Commercial Training and Enablement

Key Points 200003651 v01

• Illumina COVIDSeq Assay– RUO for surveillance of SARS-CoV-2 RNA

• Illumina COVIDSeq Assay– RUO workflow steps include cDNA synthesis, amplification of target
regions, tagmentation of amplicons, pooling and clean up of final libraries
• User-supplied reagents and equipment required for RNA extraction and Library Quantification
steps
• Library pooling should be traced carefully for sample tracking and appropriate sequencing
system recommendations
• Reference sequencing system documentation for denature and dilution recommendations
• Best practices should be followed when running this assay
• Avoid cross contamination throughout protocol
• Include No Template Control to monitor contamination
• Handle and prepare reagents as specified
• Follow protocol as written

For Research Use Only. Not for use in diagnostic procedures.

 Created by Commercial Training and Enablement
Reference Documentation 200003651 v01

Resources

Illumina COVIDSeq RUO Kits Reference Guide

• Document # 1000000126053
Illumina COVIDSeq Software Guide
• Document # 1000000128122
Illumina DRAGEN COVIDSeq Test App Guide
• Document # 1000000129548

For Research Use Only. Not for use in diagnostic

procedures.
 Created by Commercial Training and Enablement
200003651 v01

Disclaimer

This course has been developed using the Illumina

COVIDSeq RUO Kits Reference Guide.

Refer to the Illumina Support website for the most

current version of the Illumina COVIDSeq RUO Kits
Reference Guide.

For Research Use Only. Not for use in diagnostic procedures.

 200003651 v01

Questions?