IGA NovaSeq Sequencing v2.1

Overview

IGA NovaSeq Sequencing v2.1 contains preset protocols that support the setup of previously prepared libraries used with the NovaSeq instrument. These protocols can be used with the IGA Library Prep Automated v2.1 workflow. They also provide the following functions:

• File-based integration with the Hamilton robots that are used for liquid handling steps.
• Automated calculation of sample and buffer volumes.
• Automated calculation or display of reagents.
• When required, automatic step transition.

Protocol 1: Run Format (IGA)

Protocol Type = Library Prep

Next Steps Configuration

Step 1: Define Run Format (IGA)

• Master Step Name = Define Run Format (IGA v2.1)
• Step Type = No Outputs

Automations

Register Sample & Register Step Started (IGA v2.1)

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

Define Run Format - Assign Workflow and Flowcell Type to Samples & Set Next Steps to Remove from Workflow (IGA v2.1)

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

Define Run Format - Routing Script (IGA v2.1)

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

Queue

• 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	Well	Built-in
    Derived Sample	Reagent Name	Built-in
    Derived Sample	Sample Name	Built-in
    Derived Sample	Waiting	Built-in

  • Expanded View Fields

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

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	Well	Built-in
    Derived Sample	Reagent Name	Built-in
    Derived Sample	Sample Name	Built-in
    Derived Sample	Waiting	Built-in

  • Expanded View Fields

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

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  IGA WF Build Version	Text	Read Only	Default = 2.1.0.1
  Quick reference checklist: Define Run Format.	Multiline Text	Read Only	Defaults = User tasks for this page: - Below, assign 'Loading Workflow Type', 'Flowcell Type', and the NovaSeq 'Final Loading Concentration (pM)'. - Click 'Next Steps'.

• Step File Placeholders

  • Log file - Automatically attached
• Sample Table (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	Adjusted Per Sample Volume (ul)	Numeric	Read Only	Decimal Places Displayed = 2
  Derived Sample	Final Loading Concentration (pM)	Numeric Dropdown	Required Field Custom Entries	Presets 225 400 Decimal Places Displayed = 0
  Derived Sample	Flowcell Type	Text Dropdown	Required Field	Presets SP S1 S2 S4
  Derived Sample	Loading Workflow Type	Text Dropdown	Required Field	Presets NovaSeq Standard NovaSeq Xp [Remove from workflow]
  Derived Sample	Minimum Molarity (nM)	Numeric		Decimal Places Displayed = 2
  Derived Sample	Normalized Molarity (nM)	Numeric		Decimal Places Displayed = 2
  Derived Sample	Per Sample Volume (ul)	Numeric	Read Only	Decimal Places Displayed = 2
  Derived Sample	Sample Name	Built-in
  Derived Sample	Warning	Text	Read Only
  Project	Project Name	Built-in

Protocol 2: NovaSeq Standard

Protocol Type = Library Prep

Next Steps Configuration

Step 1: Make Bulk Pool for NovaSeq Standard

• Master Step Name = Make Bulk Pool for NovaSeq Standard (IGA v2.1)
• Step Type = Pooling
• Aliquot Generation = Fixed, 1
• Naming Convention = {PoolName}
• Control Types

  • PhiX v3

Automations

Register Sample & Register Step Started (IGA v2.1)

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

Pooling NovaSeq Standard - Show Warning Message (IGA v2.1)

• Trigger Location = Pooling
• Trigger Style = Automatic upon entry

Validate Inputs Flowcell Type and Number of Pools (IGA v2.1)

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

NovaSeq Make Bulk Pool - Output Plate BPP Barcode Check (Duplicates Allowed) (IGA v2.1)

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

Make Bulk Pool NovaSeq Standard - Calculate Volumes and Generate Hamilton Input File (IGA v2.1)

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

Make Bulk Pool NovaSeq Standard - Validate Hamilton Output File Name if Present, Parse Instrument ID, Set Next Steps to Advance (IGA v2.1)

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

Register Step Completed (IGA v2.1)

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

Validate Unique Indexes (IGA v2.1)

• Trigger Location = Not Used

Queue

• 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	Well	Built-in
    Derived Sample	Flowcell Type	Text Dropdown	Required Field	Presets SP S1 S2 S4
    Derived Sample	Normalized Molarity (nM)	Numeric		Decimal Places Displayed = 2
    Derived Sample	Sample Name	Built-in
    Derived Sample	Waiting	Built-in

  • Expanded View Fields

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

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	Well	Built-in
    Derived Sample	Flowcell Type	Text Dropdown	Required Field	Presets SP S1 S2 S4
    Derived Sample	Normalized Molarity (nM)	Numeric		Decimal Places Displayed = 2
    Derived Sample	Sample Name	Built-in
    Derived Sample	Waiting	Built-in

  • Expanded View Fields

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

Pooling

• Label Uniqueness = On
• Defaults

  • Sample Grouping = Group by Containers
  • Well Sort Order = Column

Placement = Enabled

• Defaults

  • Well Sort Order = Column
  • Placement Pattern = Column
• Destination Containers

  • 96 well plate

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Number of Flowcells to Sequence	Numeric	Required Field	Default = 1 Range From 1 to 10 Decimal Places Displayed = 0
  Minimum Per Sample Volume (ul)	Numeric	Required Field	Default = 2 Decimal Places Displayed = 2
  Liquid Handling Instrument ID	Text	Read Only
  Apply Correction Factors	Text Dropdown	Required Field	Presets None IDPF Custom Default = None
  Quick reference checklist: Make Bulk Pool for NovaSeq Standard. i) pre Liquid Handler	Multiline Text	Read Only	Defaults = User tasks for this page: - Select 'Number Of Flowcells' and 'Min Per Sample Volume (ul)' - 'Run' the Make Bulk Pool automation, and download resulting Hamilton Make Bulk Pool Input File
  Quick reference checklist: Make Bulk Pool for NovaSeq Standard. ii) post Liquid Handler	Multiline Text	Read Only	Defaults = User tasks for this page: - After running MakeBPP on the liquid handler, upload resulting Hamilton Output File - Click on 'Next Steps'

• Step File Placeholders

  • Calculation Log - Automatically attached
  • Hamilton Make Bulk Pool Input File - Automatically attached
  • Hamilton file log - Automatically attached
  • Hamilton Output File - Manually uploaded
  • Custom Correction Factors - Manually uploaded
  • Index Correction Log - Automatically attached
• Sample Table (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	Flowcell Type	Text Dropdown	Required Field	Presets SP S1 S2 S4
  Derived Sample	Loading Workflow Type	Text Dropdown	Required Field	Presets NovaSeq Standard NovaSeq Xp [Remove from workflow]
  Derived Sample	NaOH Volume (ul)	Numeric	Read Only	Decimal Places Displayed = 2
  Derived Sample	RSB Volume (ul)	Numeric	Read Only	Decimal Places Displayed = 2
  Derived Sample	Sample Name	Built-in
  Derived Sample	Tris-HCl Volume (ul)	Numeric	Read Only	Decimal Places Displayed = 2
  Derived Sample	Volume of Pool to Denature (ul)	Numeric	Read Only	Decimal Places Displayed = 0
  Project	Project Name	Built-in

Step 2: Dilute and Denature

• Master Step Name = Dilute and Denature (IGA v2.1)
• Step Type = Standard
• Derived Sample Generation = Fixed, 1
• Naming Convention = {InputItemName}
• Reagent Kits

  • NaOH

    • Supplier = Illumina
  • Resuspension Buffer (RSB)

    • Supplier = Illumina
  • Tris-HCl

    • Supplier = Illumina

Automations

Validate Single Input (IGA v2.1)

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

Validate Library Tube Barcode (IGA v2.1)

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

Dilute and Denature NovaSeq Standard - Generate Hamilton Library Tube Input File, Validate Run Setup, and Generate SampleSheet (IGA v2.1)

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

Dilute and Denature NovaSeq Standard - Set Next Steps to Remove from Workflow (IGA v2.1)

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

Dilute and Denature NovaSeq Standard - Routing Script (IGA v2.1)

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

Queue

• 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	Well	Built-in
    Derived Sample	Flowcell Type	Text Dropdown	Required Field	Presets SP S1 S2 S4
    Derived Sample	Sample Name	Built-in
    Derived Sample	Waiting	Built-in

  • Expanded View Fields

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

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	Well	Built-in
    Derived Sample	Flowcell Type	Text Dropdown	Required Field	Presets SP S1 S2 S4
    Derived Sample	Sample Name	Built-in
    Derived Sample	Waiting	Built-in

  • Expanded View Fields

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

Placement = Enabled

• Defaults

  • Sample Grouping = Group by Containers
  • Well Sort Order = Column
  • Placement Pattern = Column
• Destination Containers

  • Library Tube

  

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Adapter	Text	Required Field	Default = CTGTCTCTTATACACATCT
  Adapter Read 2	Text	Required Field	Default = CTGTCTCTTATACACATCT
  BaseSpace App Slug	Text Dropdown	Required Field Custom Entries	Presets Illumina-inc.bcl-convert.1.3.0 Illumina-inc.bcl-convert.1.2.0
  BaseSpace Sequence Hub Analysis Workflow	Text	Required Field	Default = TSS Connect v1.1
  BaseSpace Sequence Hub Configuration	Text Dropdown	Required Field	Presets Run Monitoring And Storage Run Monitoring Only Not Used Default = Run Monitoring And Storage
  BaseSpace Sequence Hub Project Name	Text	Required Field
  Custom Recipe Path	Text
  Enable Secondary Analysis	Toggle Switch	Required Field	Default = Yes
  Experiment Name	Text	Required Field
  Index Read 1	Numeric Dropdown	Required Field Custom Entries	Presets 10 6 8 0 Default = 10 Range From 0 to 20 Decimal Places Displayed = 0
  Index Read 2	Numeric Dropdown	Required Field Custom Entries	Presets 10 6 8 0 Default = 10 Range From 0 to 20 Decimal Places Displayed = 0
  Liquid Handling Instrument ID	Text	Read Only
  Output Folder	Text	Required Field
  Paired End	Text Dropdown	Required Field	Presets True False Default = True
  Quick reference checklist: Dilute and Denature.	Multiline Text	Read Only	Defaults = User tasks for this page: - Select NaOH/TrisHCl/RSB lots, and input run configuration - 'Run' Dilute and Denature automation, download resulting input 'Hamilton Load to Library tube file' - Click 'Next Steps' and then 'Finish Step' to move library tube through to 'AUTOMATED - NovaSeq Run'.
  Read 1 Cycles	Numeric Dropdown	Required Field Custom Entries	Presets