The MiniSeq workflow includes the following functionality.
Preconfigured protocols that support the preparation of small genome, amplicon, targeted enrichment, and RNA sequencing using low library volumes for sequencing.
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.
Master Step Name = Dilute Stock PhiX (MiniSeq v1.0)
Step Type = Standard
Derived Sample Generation = Fixed, 1
Automations
Calculate Stock PhiX and RSB Volumes
Trigger Location = Record Details
Trigger Style = Manual button
Set Next Step - Advance
Trigger Location = Record Details
Trigger Style = Automatic upon exit
Queue/Ice Bucket
Defaults
Sample Grouping = Group by Containers
Well Sort Order = Column
Record Details
Step Data (Master Step Fields)
Step 2: Denature PhiX (MiniSeq v1.0)
Master Step Name = Denature PhiX (MiniSeq v1.0)
Step Type = Standard
Derived Sample Generation = Fixed, 1
Automations
Set Next Step - Advance
Trigger Location = Record Details
Trigger Style = Automatic upon exit
Queue/Ice Bucket
Defaults
Sample Grouping = Group by Containers
Well Sort Order = Column
Record Details
Step Data (Master Step Fields)
Step 3: Dilute PhiX to Loading Concentration and Combine Library & PhiX (MiniSeq v1.0)
Master Step Name = Dilute PhiX to Loading Concentration and Combine Library & PhiX (MiniSeq v1.0)
Step Type = Pooling
Aliquot Generation = Fixed, 1
Automations
Calculate Denatured PhiX and Prechilled Hybridization Buffer Volumes
Trigger Location = Record Details
Trigger Style = Manual button
Set Next Step - Advance
Trigger Location = Record Details
Trigger Style = Automatic upon exit
Queue/Ice Bucket
Defaults
Sample Grouping = Group by Containers
Well Sort Order = Column
Pooling
Label Uniqueness = On
Defaults
Sample Grouping = Group by Containers
Record Details
Step Data (Master Step Fields)
Protocol 7: Run Setup (MiniSeq v1.0)
Protocol Type = Sequencing
Next Steps Configuration
Step 1: Run Setup (MiniSeq v1.0)
Master Step Name = Run Setup (MiniSeq v1.0)
Step Type = Standard
Derived Sample Generation = Fixed, 1
Automations
1. miniseq sample sheet
Trigger Location = Record Details
Trigger Style = Manual button
Set Next Step - Advance
Trigger Location = Record Details
Trigger Style = Automatic upon exit
Queue/Ice Bucket
Defaults
Sample Grouping = Group by Containers
Well Sort Order = Column
Placement = Enabled
Defaults
Sample Grouping = Group by Containers
Well Sort Order = Row
Record Details
Step Data (Master Step Fields)
ℹ The workflow version and step version for the routing script may vary depending on the version of the IPP.
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 = Select a Normalization Variation protocol for each sample.
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Expand
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
Resuspension Buffer (RSB)
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 = 10
Range = 10 To 400
Decimal Places Displayed = 0
Instruction Notes
Multiline Text
Read Only
Default =
- Enter in Final Molarity (nM), Final Volume (uL) and Library Molarity (nM) before clicking on calculation button.
- Add 10 uL of each 10 nM library to a new microcentrifuge tube.
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Expand
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
Resuspension Buffer (RSB)
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 = 100
Instruction Notes
Multiline Text
Read Only
Default = Enter in Final Molarity (nM), and Final Volume (uL) before clicking on calculation button.
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Expand
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
0.1 N NaOH
200 mM Tris-HCl, pH 7.0
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 - Automatically attached
Sample Table
Sample Display Default = Collapse
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
Hybridization Buffer
Supplier = Illumina; part of MiniSeq High Output Reagent Kit
ℹ The workflow version and step version for the routing script may vary depending on the version of the IPP.
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 985 µl of prechilled Hybridization Buffer to the tube of denatured library.
- The total volume is 1 ml at 5 pM.
- Vortex briefly and then centrifuge at 280 × g for 1 minute.
- Transfer 180 µl diluted library to a new microcentrifuge tube.
- Add 320 µl prechilled Hybridization Buffer.
- The total volume is 500 µl at 1.8 pM.
Loading Concentration (pM)
Numeric
Default = 1.8
Total Volume (uL)
Numeric
Default = 500
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Collapse
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
Hybridization Buffer
Supplier = Illumina; part of MiniSeq High Output Reagent Kit
ℹ The workflow version and step version for the routing script may vary depending on the version of the IPP.
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 =
- Enter in Library Pool and Final Volume before clicking on calculation button.
- Vortex briefly and then centrifuge at 280 × g for 1 minute.
- Transfer 250 µl diluted library to a new microcentrifuge tube.
- Add 250 µl prechilled Hybridization Buffer.
- Vortex briefly and then centrifuge at 280 × g for 1 minute.
- To denture the diluted library pool, place the tube in the incubator for 2 minutes. Immediately cool on ice and leave it on ice for 5 minutes.
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Expand
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
Low TE
Supplier = Supplier - Illumina; part of AmpliSeq Library Plus Kit
Default =
- Select the AmpliSeq for Illumina Panel before clicking on Calculate DNA to RNA ratio button.
- Combine equal library volumes to a 1.5 mL LoBind tube.
- User different tubes for DNA and RNA Libraries.
- Combine DNA to RNA ratio in a new tube.
Transfer Library Volume (uL)
Numeric
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Expand
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
Low TE
Supplier = Supplier - Illumina; part of AmpliSeq Library Plus Kit
Default =
- Combine Pool Libraries and 0.2 N NaOH volumes.
- Vortex briefly. Incubate at room temp for 5 mins.
- Add 10 µl 200 mM Tris-HCl, pH 7.0 to the tube. Vortex briefly.
Pool Libraries (uL)
Numeric
Default = 10
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Collapse
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
HT1
Supplier = Illumina; part of AmpliSeq Library Plus Kit
ℹ The workflow version and step version for the routing script may vary depending on the version of the IPP.
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
Presets
No
Yes
Final Volume (uL)
Numeric
Default = 1000
Instruction Notes
Multiline Text
Read Only
Default =
- Enter Loading Concentration before clicking on the calculation button.
- Add 970 uL of prechilled HT1 to denatured library pool.
- Dilute to loading concentration by combining Diluted Denatured Libraries and Prechilled HT1 volumes.
Loading Concentration (pM)
Numeric
Default = 1.8
Range = 1.1 To 1.9
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Expand
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {PoolName}
Reagent Kits
Low TE
Supplier = Supplier - Illumina; part of AmpliSeq Library Plus Kit
Default =
- Select the AmpliSeq for Illumina Panel before clicking on Calculate DNA to RNA ratio button.
- Combine equal library volumes to a 1.5 mL LoBind tube.
- User different tubes for DNA and RNA Libraries.
- Combine DNA to RNA ratio in a new tube.
Transfer Library Volume (uL)
Numeric
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Expand
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
Low TE
Supplier = Supplier - Illumina; part of AmpliSeq Library Plus Kit
Default =
- Combine Pool Libraries and 0.2 N NaOH volumes.
- Vortex briefly. Incubate at room temp for 5 mins.
- Add 10 µl 200 mM Tris-HCl, pH 7.0 to the tube. Vortex briefly.
Pool Libraries (uL)
Numeric
Default = 10
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Collapse
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
HT1
Supplier = Illumina; part of AmpliSeq Library Plus Kit
ℹ The workflow version and step version for the routing script may vary depending on the version of the IPP.
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
Presets
No
Yes
Final Volume (uL)
Numeric
Default = 1000
Instruction Notes
Multiline Text
Read Only
Default =
- Enter Loading Concentration before clicking on the calculation button.
- Add 970 uL of prechilled HT1 to denatured library pool.
- Dilute to loading concentration by combining Diluted Denatured Libraries and Prechilled HT1 volumes.
Loading Concentration (pM)
Numeric
Default = 1.8
Range = 1.1 To 1.9
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Expand
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
Resuspension Buffer (RSB)
Control Types
PhiX v3
Supplier = Illumina
Catalog Number = FC-110-3001
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 = 25
Instruction Notes
Multiline Text
Read Only
Default = Enter Stock PhiX (nM), Final PhiX Molarity (nM), and Final Volume (uL) before clicking on calculation button.
RSB (uL)
Numeric
Decimal Places Displayed = 0
Stock PhiX Molarity (nM)
Numeric
Default = 10
Stock PhiX Volume (uL)
Numeric
Decimal Places Displayed = 0
Step File Placeholders
Log - Automatically attached
Sample Table
Sample Display Default = Collapse
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {InputItemName}
Reagent Kits
0.1 N NaOH
200 mM Tris-HCl, pH 7.0
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 - Automatically attached
Sample Table
Sample Display Default = Collapse
Well Sort Order = Column
Table Columns - Global Fields
Naming Convention = {PoolName}
Reagent Kits
Hybridization Buffer * Supplier = Illumina; part of MiniSeq High Output Reagent Kit * Catalog Number = 75 cycles - FC-420-1001; 150 cycles - FC-420-1002; 300 cycles - FC-420-1003 * Website = www.illumina.com
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
Decimal Places Displayed = 0
Final Denatured PhiX Molarity (pM)
Numeric
Default = 1.8
Final Volume (uL)
Numeric
Default = 500
Instruction Notes
Multiline Text
Read Only
Default =
- Enter Denatured PhiX Molarity (pM), Final Denatured PhiX Molarity (pM) and Final Volume (uL) before clicking on calculation button.
- Add Denatured PhiX and Prechilled Hybridization Buffer volumes too dilute the denatured PhiX.
- Invert to mix and then centrifuge at 280 × g for 1 minute. - Combine 5 uL of denatured and diluted PhiX control with 500 uL of denatured and diluted library pool.
Default = Add 500 uL of prepared libraries to reagent cartridge.
Local Run Manager Analysis Id
Text
Manifest File Name
Text
Module
Text
Output Folder Location
Text
Read 1 Cycles
Numeric
Required Field
Range = 0 To 151
Read 2 Cycles
Numeric
Required Field
Range = 0 To 151
Read Type
Text Dropdown
Required Field
Presets
Paired End Read
Single Read
Workflow
Text
Default = GenerateFASTQ
Step File Placeholders
Log - Automatically attached
LRM Sample Sheet - Automatically attached
Manual Sample Sheet - Automatically attached
Sample Table
Sample Display Default = Collapse
Well Sort Order = Column
Table Columns - Global Fields
Field Name
Field Type
Options
Additional Options and Dropdown Items
Comment
Multiline Text
Instruction Notes
Multiline Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
Final Molarity (nM)
Numeric
Default = 10
Range = 1 To 10
Decimal Places Displayed = 0
Final Volume (uL)
Numeric
Field Name
Field Type
Options
Additional Options and Dropdown Items
Final Molarity (nM)
Numeric
Default = 1
Final Volume (uL)
Numeric
Field Name
Field Type
Options
Additional Options and Dropdown Items
Instruction Notes
Multiline Text
Read Only
Default =
- Combine 5 uL of 1 nM Library Pool and 5 uL of 0.1 N NaOH in a microcentridge tube.
- Vortex briefly and then centrifuge at 280 × g for 1 minute.
- Incubate at room temperature for 5 minutes.
- Add 5 µl 200 mM Tris-HCl, pH 7.0. Vortex briefly and then centrifuge at 280 × g for 1 minute.
Field Name
Field Type
Options
Additional Options and Dropdown Items
Add PhiX Control
Text Dropdown
Required Field
Presets
No
Yes
Default = No
Instruction Notes
Multiline Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
Add PhiX Control
Text Dropdown
Required Field
Presets
No
Yes
Default = No
Instruction Notes
Multiline Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
Final Molarity (nM)
Numeric
Default = 2
Final Volume (uL)
Numeric
Field Name
Field Type
Options
Additional Options and Dropdown Items
AmpliSeq for Illumina Panel
Text Dropdown
Required Field
Presets
Childhood Cancer Panel
Comprehensive Panel
Focus Panel
Myeloid Panel
Default = Childhood Cancer Panel
DNA to RNA Ratio
Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
0.2 N NaOH
Numeric
Default = 10
0.2 N NaOH Prep Date
Date
Field Name
Field Type
Options
Additional Options and Dropdown Items
200 mM Tris-HCl, pH 7.0
Numeric
Default = 10
Add PhiX Control
Text Dropdown
Field Name
Field Type
Options
Additional Options and Dropdown Items
AmpliSeq for Illumina Panel
Text Dropdown
Required Field
Presets
Childhood Cancer Panel
Comprehensive Panel
Focus Panel
Myeloid Panel
Default = Childhood Cancer Panel
DNA to RNA Ratio
Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
0.2 N NaOH
Numeric
Default = 10
0.2 N NaOH Prep Date
Date
Field Name
Field Type
Options
Additional Options and Dropdown Items
200 mM Tris-HCl, pH 7.0
Numeric
Default = 10
Add PhiX Control
Text Dropdown
Field Name
Field Type
Options
Additional Options and Dropdown Items
Final PhiX Molarity (nM)
Numeric
Default = 4
Final PhiX Volume (uL)
Numeric
Field Name
Field Type
Options
Additional Options and Dropdown Items
Instruction Notes
Multiline Text
Read Only
Default =
- Combine 5uL of 4 nM PhiX and 5 uL of 0.1 N NaOH.
- Vortex briefly and then pulse centrifuge.
- Incubate at room temperature for 5 minutes.
- Add 5 µl 200 mM Tris-HCl, pH 7.0.
- Vortex briefly and then centrifuge at 280 × g for 1 minute.
