The Illumina COVIDSeq Workflow workflow includes the following functionality:
Preset Illumina COVIDSeq Workflow protocols that track samples through the quantitative detection of SARS-CoV-2 RNA from nasopharyngeal (NP), oropharyngeal (OP), and nasal swab as well as mid-turbinate specimens collected from individuals who meet COVID-19 clinical or epidemiological criteria.
Automated calculation of sample and buffer volumes.
Automated calculation or display of reagents at every step in the protocol.
Automatic step transition when required.
Automatic placement of samples when necessary.
Automated assignment of QC Pass/Fail, based on user-selected threshold values.
Sample preparation for use on the NovaSeq 6000 Sequencing System.
Step Data Heading = WARNING: It might take several minutes to parse the TSV Run Report
Step File Placeholders
Naming Convention = {InputItemName}
Reagent Kits
Illumina COVIDSeq Positive Control HT
Supplier = Illumina
Website =
Illumina COVIDSeqTest Box 4
Supplier = Illumina
Catalog Number = 20043436
Sample Table (Column Headers)
Category
Field Name
Field Type
Options
Additional Options and Dropdown Items
Container
Container Name
Built-in
Container
LIMS ID (Container)
Built-in
Step File Placeholders
Log File - Automatically attached
Sample Table
Sample Display Default = Collapse
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
QIAmp Viral RNA Mini kit
Supplier = Qiagen
Catalog Number = (50) - 52904; (250) - 52906
Website =
Sample Table (Column Headers)
Category
Field Name
Field Type
Options
Additional Options and Dropdown Items
Container
Container Name
Built-in
Container
LIMS ID (Container)
Built-in
AW1 Concentrate (mL)
Numeric
AW2 Concentrate (mL)
Numeric
Buffer AVL (mL)
Numeric
Decimal Places Displayed = 0
Carrier RNA-Buffer AVE (uL)
Numeric
Decimal Places Displayed = 0
Elution Incubation End Time
Numeric
Elution Incubation Start Time
Numeric
EtOH for AW1 (mL)
Numeric
EtOH for AW2 (mL)
Numeric
Instruction Notes
Multiline Text
Read Only
Default =
Enter in No. of Preps before clicking on "Calculate AW1, AW2, AVL Reagent Volumes".
Elute in 30 µl Buffer AVE instead of 60 µl. Incubate elution for at least 1 minute.
No. of Preps
Numeric Dropdown
Required Field
Custom Entries
Presets
250
50
Total Samples
Numeric
Default = 0
Step File Placeholders
Log File - Automatically attached
Sample Table
Sample Display Default = Collapse
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
Illumina COVIDSeq Test Box 5 – 3072 Samples
Supplier = Illumina
Catalog Number = 20043648
Website =
Sample Table (Column Headers)
Category
Field Name
Field Type
Options
Additional Options and Dropdown Items
Container
Container Name
Built-in
Container
LIMS ID (Container)
Built-in
Default =
Add 8.5 µl EPH3 HT to each well.
Add 8.5 µl eluted sample to each well.
Seal and shake at 1600 rpm for 1 minute.
Centrifuge at 1000 × g for 1 minute.
Thermal Cycler Program Notes
Text
Default = COVIDSeq Anneal
Step File Placeholders
Log File - Automatically attached
Sample Table
Sample Display Default = Collapse
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
Illumina COVIDSeq Test Box 6 – 3072 Samples
Supplier = Illumina
Catalog Number = 20043647
Website =
Sample Table (Column Headers)
Category
Field Name
Field Type
Options
Additional Options and Dropdown Items
Container
Container Name
Built-in
Container
LIMS ID (Container)
Built-in
Instruction Notes
Multiline Text
Read Only
Default = Add 8 µl First Strand cDNA Master Mix to each well of the CDNA1 plate.
RVT (uL)
Numeric
Total Samples
Numeric
Required Field
Default = 0
Thermal Cycler Program
Text
Default = COVIDSeq FSS
Step File Placeholders
Log File - Automatically attached
Sample Table
Sample Display Default = Collapse
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
Illumina COVIDSeq Test Box 5 – 3072 Samples
Supplier = Illumina
Catalog Number = 20043648
Website =
Illumina COVIDSEQ Test Box 7 – 3072 Samples
Supplier = Illumina
Catalog Number = 20043439
Sample Table (Column Headers)
Category
Field Name
Field Type
Options
Additional Options and Dropdown Items
Container
Container Name
Built-in
Container
LIMS ID (Container)
Built-in
Decimal Places Displayed = 0
CPP2 HT for COVIDSeq PCR 2 Master Mix (uL)
Numeric
Decimal Places Displayed = 0
Instruction Notes
Multiline Text
Read Only
Default = Add 20 µl COVIDSeq PCR 1 Master Mix to each well of the COV1 plate corresponding to each well of the
CDNA1 plate. Add 5 µl first strand cDNA synthesis from each well of the CDNA1 plate to the corresponding well of the COV1
plate.
Add 20 µl COVIDSeq PCR 2 Master Mix to each well of the COV2 plate corresponding to each well of the
CDNA1 plate. Add 5 µl first strand cDNA synthesis from each well of the CDNA1 plate to the corresponding well of the COV2
plate.
IPM HT for COVIDSeq PCR 1 Master Mix (uL)
Numeric
Decimal Places Displayed = 0
IPM HT for COVIDSeq PCR 2 Master Mix (uL)
Numeric
Decimal Places Displayed = 0
Nuclease-free water for COVIDSeq PCR 1 Master Mix (uL)
Numeric
Decimal Places Displayed = 0
Nuclease-free water for COVIDSeq PCR 2 Master Mix (uL)
Numeric
Decimal Places Displayed = 0
Thermal Cycler Program
Text
Default = COVIDSeq PCR
Total Samples
Numeric
Default = 0
Step File Placeholders
Log File - Automatically attached
Sample Table
Sample Display Default = Collapse
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
Illumina COVIDSEQ Test Box 7 – 3072 Samples
Supplier = Illumina
Catalog Number = 20043439
Website =
Illumina COVIDSeqTest Box 3 – 3072 Samples
Supplier = Illumina
Catalog Number = 20043646
Sample Table (Column Headers)
Category
Field Name
Field Type
Options
Additional Options and Dropdown Items
Container
Container Name
Built-in
Container
LIMS ID (Container)
Built-in
Decimal Places Displayed = 0
Instruction Notes
Multiline Text
Read Only
Default = Add 30 µl Tagmentation Master Mix to each well in TAG1 plate.
IDT for Illumina Nextera DNA UD Indexes Set B for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000
IDT for Illumina Nextera DNA UD Indexes Set C for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000
IDT for Illumina Nextera DNA UD Indexes Set D for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000
Illumina DNA-RNA UD Indexes Set A B C D Tagmentation
Illumina DNA-RNA UD Indexes Set A Tagmentation
Illumina DNA-RNA UD Indexes Set B Tagmentation
Illumina DNA-RNA UD Indexes Set C Tagmentation
Illumina DNA-RNA UD Indexes Set D Tagmentation
Instruction Notes
Multiline Text
Read Only
Default = Add 40 µl PCR Master Mix to each well.
Nuclease-free water (uL)
Numeric
Thermal Cycler Program
Text
Default = COVIDSeq TAG PCR
Total Samples
Numeric
Default = 0
Step File Placeholders
Log File - Automatically attached
Sample Table
Sample Display Default = Collapse
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {PoolName}
Reagent Kits
Illumina COVIDSeqTest Box 3 – 3072 Samples
Supplier = Illumina
Catalog Number = 20043646
Website =
Sample Table (Column Headers)
Category
Field Name
Field Type
Options
Additional Options and Dropdown Items
Container
Container Name
Built-in
Container
LIMS ID (Container)
Built-in
Well Sort Order = Column
Destination Containers
1x8 Strip Tube
96 well plate
Default =
Enter in Total Pooled ITB Tubes to Calculate ITB (uL) volume.
Transfer 50 µl supernatant from each Pooled ITB tube to a new microcentrifuge tube.
ITB (uL)
Numeric
Total Pooled ITB Tubes
Numeric
Step File Placeholders
Log File - Automatically attached
Sample Table
Sample Display Default = Collapse
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
Sample Table (Column Headers)
Category
Field Name
Field Type
Options
Additional Options and Dropdown Items
Container
Container Name
Built-in
Container
LIMS ID (Container)
Built-in
Default = Concentration
Criteria 1 - Threshold Value
Numeric
Default = 2
Criteria 2 - Operator
Text
Default = <>=
Criteria 2 - Source Data Field
Text
Default = Concentration
Criteria 2 - Threshold Value
Numeric
Default = 30
Step File Placeholders
Log - Automatically attached
QC Log File - Automatically attached
QC Result File - Manually uploaded
Upload File - Manually uploaded
Sample Table
Enable QC Flags = Yes
Sample Display Default = Expand
Well Sort Order = Column
File Column Options
File Column Display = Show
File Attachment Method = Manual
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
Illumina COVIDSeqTest Box 3 – 3072 Samples
Supplier = Illumina
Catalog Number = 20043646
Website =
Sample Table (Column Headers)
Category
Field Name
Field Type
Options
Additional Options and Dropdown Items
Container
Container Name
Built-in
Container
LIMS ID (Container)
Built-in
Instruction Notes
Multiline Text
Default = Enter Final Volume (uL) then click on "Calculate Sample & RSB Volumes" for dilution volumes.
Default = For each sample, add 140 µl patient sample, dilution 3 CPC HT, or ELB HT (no template control) to a new 1.7 ml microcentrifuge tube.
Field Name
Field Type
Options
Additional Options and Dropdown Items
% EtOH
Numeric
Default = 96
Range = 96 To 100
% EtOH Prep Date
Date
Field Name
Field Type
Options
Additional Options and Dropdown Items
COVIDSeq Anneal Program Notes
Multiline Text
Read Only
Default =
Choose the preheat lid option
Set the reaction volume to 17 µl
65°C for 3 minutes
Hold at 4°C
Instruction Notes
Multiline Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
COVIDSeq FSS Program Notes
Multiline Text
Read Only
Default =
Choose the preheat lid option
Set the reaction volume to 25 µl
25°C for 5 minutes
50°C for 10 minutes
80°C for 5 minutes
Hold at 4°C
FSA (uL)
Numeric
Field Name
Field Type
Options
Additional Options and Dropdown Items
COVIDSeq PCR Notes
Multiline Text
Read Only
Default =
Choose the preheat lid option
Set the reaction volume to 25 µl
98°C for 3 minutes
35 cycles of:
98°C for 15 seconds
65°C for 5 minutes
Hold at 4°C
CPP1 HT for COVIDSeq PCR 1 Master Mix (uL)
Numeric
Field Name
Field Type
Options
Additional Options and Dropdown Items
COVIDSeq TAG Program Notes
Multiline Text
Read Only
Default =
Choose the preheat lid option
Set the reaction volume to 50 µl
55°C for 5 minutes
Hold at 10°C
eBLT (uL)
Numeric
Field Name
Field Type
Options
Additional Options and Dropdown Items
COVIDSeq TAG PCR Program Notes
Multiline Text
Read Only
Default =
Choose the preheat lid option and set to 100°C
Set the reaction volume to 50 µl
72°C for 3 minutes
98°C for 3 minutes
7 cycles of:
98°C for 20 seconds
60°C for 30 seconds
72°C for 1 minute
72°C for 3 minutes
Hold at 10°C
EPM HT (uL)
Numeric
Field Name
Field Type
Options
Additional Options and Dropdown Items
80% EtOH Date
Date
Instruction Notes
Multiline Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
Criteria 1 - Operator
Text
Default = >=
Criteria 1 - Source Data Field
Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
Dilution Factor
Text
Read Only
Default = 1:10
Final Volume (uL)
Numeric
Field Name
Field Type
Options
Additional Options and Dropdown Items
Criteria 1 - Operator
Text
Default = >=
Criteria 1 - Source Data Field
Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
Final Molarity (nM)
Numeric
Default = 4
Final Volume (uL)
Numeric
Field Name
Field Type
Options
Additional Options and Dropdown Items
Instruction Notes
Multiline Text
Read Only
Default =
For each set of 384 samples, combine 25 µl of each normalized pool containing index adapter set A, B, C, D in a new microcentrifuge tube. Do not combine pools with the same index adapter set. This step produces a final pool of 384 samples for one flow cell lane.
For each flow cell lane, dilute each 384 sample pool with RSB HT as follows. These steps produce 30 µl 0.5 nM pooled library.
Combine 3.75 µl library from the 384-sample pool with 26.25 µl RSB.
Vortex, and then centrifuge briefly. The resulting final loading concentration is 100 pM.
