MiniSeq v1.0

Overview

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.

Protocol 1: Select Normalization Variation (MiniSeq v1.0)

Protocol Type = Sequencing

Next Steps Configuration

Step 1: Select Normalization Variation (MiniSeq v1.0)

• Master Step Name = Select Normalization Variation (MiniSeq v1.0)
• Step Type = No Outputs

Automations

Set Next Step - Remove

• Trigger Location = Record Details
• Trigger Style = Automatic upon exit

Routing Script for Normalization Variation

• Trigger Location = Step
• Trigger Style = Automatic upon exit

ℹ The workflow version and step version for the routing script may vary depending on the version of the IPP.

Set Next Step - Advance

• Trigger Location = Not Used

Queue/Ice Bucket

• Defaults

  • Sample Grouping = Group by Containers
  • Well Sort Order = Column
• 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
  Container	Well	Built-in
  Derived Sample	Sample Name	Built-in
  Derived Sample	Waiting	Built-in
  Project	Project Name	Built-in

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Comment	Multiline Text
  Instruction Notes	Multiline Text	Read Only	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

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	Container Name	Built-in
    Container	LIMS ID (Container)	Built-in
    Container	Well	Built-in
    Derived Sample	Sample Name	Built-in
    Derived Sample	Normalization Variation	Text Dropdown	Required Field	Presets Standard Normalization Bead-Based Normalization AmpliSeq for Illumina Normalization AmpliSeq Library Equalizer for Illumina Normalization Default = Standard Normalization
    Project	Project Name	Built-in

Protocol 2: Standard Normalization (MiniSeq v1.0)

Protocol Type = Sequencing

Next Steps Configuration

Step 1: Create Normalized Library Pool (MiniSeq v1.0)

• Master Step Name = Create Normalized Library Pool (MiniSeq v1.0)
• Step Type = Standard
• Derived Sample Generation = Fixed, 1
• Naming Convention = {InputItemName}
• Reagent Kits

  • Resuspension Buffer (RSB)

Automations

Calculate Library & RSB Volumes - MiniSeq

• 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
• 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
  Container	Well	Built-in
  Derived Sample	Sample Name	Built-in
  Derived Sample	Waiting	Built-in
  Project	Project Name	Built-in

Record Details

• Step Data (Master Step Fields)

  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		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

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	Container Name	Built-in
    Container	LIMS ID (Container)	Built-in
    Container	Well	Built-in
    Derived Sample	Final Molarity (nM)	Numeric
    Derived Sample	Library Molarity (nM)	Numeric	Required Field
    Derived Sample	Library (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	RSB (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in

Step 2: Dilute Library Pool (MiniSeq v1.0)

• Master Step Name = Dilute Library Pool (MiniSeq v1.0)
• Step Type = Standard
• Derived Sample Generation = Fixed, 1
• Naming Convention = {InputItemName}
• Reagent Kits

  • Resuspension Buffer (RSB)

Automations

Copy Library Pool Molarity

• Trigger Location = Record Details
• Trigger Style = Automatic upon entry

Calculate Library Pool & RSB Volumes - MiniSeq

• 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
• 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
  Container	Well	Built-in
  Derived Sample	Sample Name	Built-in
  Derived Sample	Waiting	Built-in
  Project	Project Name	Built-in

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Final Molarity (nM)	Numeric		Default = 1
  Final Volume (uL)	Numeric		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

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	Container Name	Built-in
    Container	LIMS ID (Container)	Built-in
    Container	Well	Built-in
    Derived Sample	Library Pool (nM)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Library Pool (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	RSB (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in

Step 3: Denature Library Pool (MiniSeq v1.0)

• Master Step Name = Denature Library Pool (MiniSeq v1.0)
• Step Type = Standard
• Derived Sample Generation = Fixed, 1
• Naming Convention = {InputItemName}
• Reagent Kits

  • 0.1 N NaOH
  • 200 mM Tris-HCl, pH 7.0

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
• 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
  Container	Well	Built-in
  Derived Sample	Sample Name	Built-in
  Derived Sample	Waiting	Built-in
  Project	Project Name	Built-in

Record Details

• Step Data (Master Step Fields)

  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.

• Step File Placeholders

  • Log - Automatically attached
• Sample Table

  • Sample Display Default = Collapse
  • Well Sort Order = Column
  • Table Columns - Global Fields

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	Container Name	Built-in
    Container	LIMS ID (Container)	Built-in
    Container	Well	Built-in
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in

Step 4: Dilute to Loading Concentration (MiniSeq v1.0)

• Master Step Name = Dilute to Loading Concentration (MiniSeq v1.0)
• Step Type = Standard
• Derived Sample Generation = Fixed, 1
• Naming Convention = {InputItemName}
• 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

Automations

Set Next Step - Advance

• Trigger Location = Record Details
• Trigger Style = Automatic upon exit

Routing Script for MiniSeq

• Trigger Location = Step
• Trigger Style = Automatic upon exit

ℹ The workflow version and step version for the routing script may vary depending on the version of the IPP.

Queue/Ice Bucket

• Defaults

  • Sample Grouping = Group by Containers
  • Well Sort Order = Column
• 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
  Container	Well	Built-in
  Derived Sample	Sample Name	Built-in
  Derived Sample	Waiting	Built-in
  Project	Project Name	Built-in

Record Details

• Step Data (Master Step Fields)

  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	Read Only	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

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	Container Name	Built-in
    Container	LIMS ID (Container)	Built-in
    Container	Well	Built-in
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in

Protocol 3: Bead-Based Normalization (MiniSeq v1.0)

Protocol Type = Sequencing

Next Steps Configuration

Step 1: Dilute Library Pool to Loading Concentration and Denature - Bead Based (MiniSeq v1.0)

• Master Step Name = Dilute Library Pool to Loading Concentration and Denature - Bead Based (MiniSeq v1.0)
• Step Type = Standard
• Derived Sample Generation = Fixed, 1
• Naming Convention = {InputItemName}
• 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

Automations

Calculate Prechilled Hybridization Buffer Volumes - MiniSeq

• Trigger Location = Record Details
• Trigger Style = Manual button

Set Next Step - Advance

• Trigger Location = Record Details
• Trigger Style = Automatic upon exit

Routing Script for MiniSeq

• Trigger Location = Step
• Trigger Style = Automatic upon exit

ℹ The workflow version and step version for the routing script may vary depending on the version of the IPP.

Queue/Ice Bucket

• Defaults

  • Sample Grouping = Group by Containers
  • Well Sort Order = Column
• 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
  Container	Well	Built-in
  Derived Sample	Sample Name	Built-in
  Derived Sample	Waiting	Built-in
  Project	Project Name	Built-in

Record Details

• Step Data (Master Step Fields)

  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	Read Only	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

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	Container Name	Built-in
    Container	LIMS ID (Container)	Built-in
    Container	Well	Built-in
    Derived Sample	Final Volume (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Library Pool (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Prechilled Hybridization Buffer (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in

Protocol 4: AmpliSeq for Illumina Normalization (MiniSeq v1.0)

Protocol Type = Sequencing

Next Steps Configuration

Step 1: Dilute Libraries - AmpliSeq (MiniSeq v1.0)

• Master Step Name = Dilute Libraries - AmpliSeq (MiniSeq v1.0)
• Step Type = Standard
• Derived Sample Generation = Fixed, 1
• Naming Convention = {InputItemName}
• Reagent Kits

  • Low TE

    • Supplier = Supplier - Illumina; part of AmpliSeq Library Plus Kit
    • Catalog Number = 24 - 20019101; 96 - 200191102; 384 - 200191103
    • Website = www.illumina.com

Automations

Copy Final Molarity

• Trigger Location = Record Details
• Trigger Style = Automatic upon entry

Calculate Starting Molarity and Library & Low TE Volumes - MiniSeq

• 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
• 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
  Container	Well	Built-in
  Derived Sample	Sample Name	Built-in
  Derived Sample	Waiting	Built-in
  Project	Project Name	Built-in

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Final Molarity (nM)	Numeric		Default = 2
  Final Volume (uL)	Numeric		Default = 50
  Instruction Notes	Multiline Text	Read Only	Default = Enter in the concentration and average library size before clicking on the calculation button.

• Step File Placeholders