Configuration

The Illumina MiSeq Integration Package v8.3.0 supports the integration of Clarity LIMS to Illumina MiSeq Sequencing Systems.

The integration allows for automated tracking of an Illumina sequencing run in Clarity LIMS. This capability includes tracking sequencing run status, generating run report, and capturing and parsing run statistics. In addition, this integration provides automated generation of a sample sheet file for use with the MiSeq Control Software (MCS) and Local Run Manager (LRM).

For instructions on user interaction for each step, validating and troubleshooting the Illumina MiSeq Integration Package, refer to MiSeq Integration v8.3.0 User Interaction, Validation and Troubleshooting.

Compatibility

• The MiSeq Sequencing v3.2 workflow is compatible with MiSeq Integration Package v8.2.0 and v8.3.0.

• MiSeq Integration v8.2.0 and above is only required for MiSeq Control Software (MCS) v4.0. For MCS v3.1 or earlier, do not upgrade to MiSeq Integration v8.2.0 or above. This upgrade breaks the integration.

Prerequisites and Assumptions

Before samples are assigned to the MiSeq Sequencing v3.2 workflow, make sure that the following prerequisites are completed:

• Samples have been accessioned into Clarity LIMS.

• Samples have been run through QC and library prep.

• Samples have been normalized, and the value is captured in a field called Normalized Molarity (nM).

For more information on sample accessioning, refer to Sample Accessioning and Upload and Modify Samples in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.

Samples can be assigned to the MiSeq Sequencing v3.2 workflow automatically using a routing script or manually from the Projects & Samples dashboard. Refer to Assign and Process Samples in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.

Workflows, Protocols, and Steps

The Illumina MiSeq Integration includes the MiSeq Sequencing v3.2 workflow, which contains a single protocol of the same name.

Step 1: Library Pooling (MiSeq v3.2)

In this step, the lab scientist manually places libraries into pools in the Clarity LIMS Placement screen.

Set Next Step - Advance Automation

This automation advances samples to the next step in the protocol. The automation is automatically triggered on exit of the Record Details screen.

The default command line is as follows.

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-0extensions.jar -i {stepURI:v2} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t false \
-h false \
-exp 'nextStep = ::ADVANCE::' \
-log {compoundOutputFileLuid0}"

Validate Maximum Number of Samples Automation

This automation checks the maximum number of samples that is allowed in a single pool. The default maximum for Illumina MiSeq Control Software (MCS) is 1536 samples. The automation is automatically triggered on entry of the Pooling screen.

The default command line is as follows.

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:validateSampleCount -min 1 -max 1536"

Master Step Fields

The following field is configured on the Library Pooling (MiSeq v3.2) master step. The field displays on the Record Details screen at run time.

Library Pooling (MiSeq v3.2) Master Step Field Configuration

Field Name	Field Type	Field Constraints/Options	Preset Values/Additional Options and Drop-down Items
Comment	Multiline Text

Global Fields

The following table lists the global fields that are displayed on the Queue and Ice Bucket screens of the Library Pooling (MiSeq v3.2) step. Most fields display in expanded view only.

Global Field Configuration (Submitted Sample)

Field Name	Field Type	Field Constraints/Options	Preset Values/Additional Options and Drop-Down Items
Application	Text Dropdown	Custom Entries	Presets TruSeq mRNA sequencing TruSeq DNA sequencing (large genome de novo) TruSeq DNA sequencing (large genome re-seq) TruSeq DNA sequencing (small genome de novo) TruSeq DNA sequencing (small genome re-seq) Nextera DNA sequencing TruSeq Custom Amplicon sequencing ChIP-sequencing Exome sequencing Mate pair sequencing Small RNA sequencing
Pooling	Text Dropdown	Custom Entries	Presets Yes No
Read Length	Text
Sequencing Coverage	Text
Sequencing Method	Text Dropdown	Custom Entries	Presets Single Read Paired End Read Indexed Single Read Indexed Paired End Read

Global Field Configuration (Derived Sample)

Field Name	Field Type	Field Constraints/Options	Preset Values/Additional Options and Drop-Down Items
Normalized Molarity (nM)	Numeric		Decimal places displayed = 2

Step 2: Denature and Dilute (MiSeq v3.2)

In this step, pooled libraries are denatured and diluted, and then placed into the reagent cartridge loaded into the MiSeq instrument.

Validate Single Input Automation

This automation checks that there is only one pooled input in the step. The automation is automatically triggered when starting the step.

The default command line is as follows.

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:validateSampleCount -min 1 -max 1"

Validate Run Setup and Generate MiSeq SampleSheet Automation

This automation validates the information entered on the Record Details screen, generates the sample sheet (refer to Sample Sheet Generation), and attaches the sample sheet to the Denature and Dilute step.

Select a button on the Record Details screen to trigger this automation.

Validation Rules

• The Experiment Name custom field cannot exceed 40 characters.

• The Experiment Name field can only contain letters, numbers, periods, nonconsecutive spaces, and the following special characters: `~!@#%-_}{.

• If Starling is selected as the variant caller, the Export to gVCF field must be set to Yes.

• If the Read 2 Cycles field is empty or 0, the following rules apply:

  • The Custom Primers field cannot have a selection that contains Read 2.

  • The Adapter Read 2 field cannot have a value.

  • The UMI - Read 2 Length field and the UMI - Read 2 Start From Cycle field cannot have values.

• The UMI - Read 1 Length field and the UMI - Read 1 Start From Cycle field cannot be used independently.

• The UMI - Read 2 Length field and the UMI - Read 2 Start From Cycle field cannot be used independently.

• If the UMI - Read 2 Length field is greater than 0, the UMI - Read 1 Length field must be greater than 0.

• The Adapter Read 1 and Adapter Read 2 fields can only contain ACGT+ characters. The adapter sequence cannot start or end with + or contain more than one +.

• For DNA Enrichment analysis, if the Variant Caller value is Somatic, the Variant Frequency Percentage must be between 1 and 100.

• For DNA Amplicon analysis, if the Variant Caller value is Somatic, the Variant Frequency Percentage field must contain a value between 0.05 and 30.

• If the Aligner value is BWA or TruSeq Amplicon Aligner, an error occurs when the Workflow value is not equal to DNA Amplicon.

• If the Aligner value is BWA-MEM or BWA-Backtrack Legacy, an error occurs when the Workflow value is equal to DNA Amplicon.

• If the Variant Caller value is Starling or GATK, an error occurs when the Workflow value is equal to DNA Amplicon.

• If the Variant Caller value is Somatic, an error occurs when the Workflow value is not equal to DNA Enrichment or DNA Amplicon.

• If the Variant Caller value is Germline, an error occurs when the Workflow value is not equal to DNA Amplicon.

The default command line is as follows.

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
script:evaluateDynamicExpression \
-exp {udf:Validation Script 1} {udf:Validation Script 2}'\
-log {compoundOutputFileLuid3} \
-t true \
&& /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} \
script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/MiSeqSamplesheetv3.csv \
-o {compoundOutputFileLuid1}.csv \
-q true \
-destLIMSID {compoundOutputFileLuid1} \
-l {compoundOutputFileLuid2}"

There is an 8000 character limit in the Oracle database used for the automation storage. Because of this character limit, the automation splits and saves the validation expressions to the Validation Script 1 and Validation Script 2. These fields are configured as master step fields.

Validation Script 1 Command Line

if (step.::Experiment Name::.length() > 40) { fail(::Experiment Name cannot exceed 40 characters.::) }; if (!step.::Experiment Name::.matches(::^(?!.*[ ]{2})[a-zA-Z0-9-_`.~!#@%{ }]+::)) { fail(::Experiment Name contains prohibited characters. Allowed characters are letters, numbers, periods, non-consecutive spaces and the following special characters: `~!@#%-_}{::) }; if (step.::Workflow:: == ::Resequencing:: && step.::Variant Caller:: == ::Starling:: && (!step.hasValue(::Export to gVCF::) || step.::Export to gVCF:: != ::Yes::)) { fail(::Export to gVCF must be set to Yes for Starling variant caller.::) }; if (step.::Workflow:: == ::DNA Enrichment:: && step.::Variant Caller:: == ::Somatic:: && (!step.hasValue(::Variant Frequency Percentage::) || step.::Variant Frequency Percentage:: > 100 || step.::Variant Frequency Percentage:: < 1)) { fail(::In the Variant Frequency Percentage field, please enter values between 1 and 100.::) }; if (step.::Workflow:: == ::DNA Amplicon:: && step.::Variant Caller:: == ::Somatic:: && (!step.hasValue(::Variant Frequency Percentage::) || step.::Variant Frequency Percentage:: > 30 || step.::Variant Frequency Percentage:: < 0.05)) { fail(::In the Variant Frequency Percentage field, please enter values between 0.05 and 30.::) }; if (!step.hasValue(::Read 2 Cycles::) || step.::Read 2 Cycles:: == 0) { if (step.::Custom Primers::.contains(::Read 2::)) { fail(::Custom Primers setting selected is invalid and can only be used in a Paired-End run.::) }; if (step.hasValue(::Adapter Read 2::)) { fail(::Adapter Read 2 is only applicable for a Paired-End run.::) }; if (step.hasValue(::UMI - Read 2 Length::) || step.hasValue(::UMI - Read 2 Start From Cycle::)) { fail(::UMI - Read 2 Length and UMI - Read 2 Start From Cycle are only applicable for a Paired-End run.::) }; }; if (step.hasValue(::UMI - Read 1 Length::) && !step.hasValue(::UMI - Read 1 Start From Cycle::)) { fail(::UMI - Read 1 Start From Cycle must be greater than 0 if UMI - Read 1 Length is greater than 0.::) }; if (!step.hasValue(::UMI - Read 1 Length::) && step.hasValue(::UMI - Read 1 Start From Cycle::)) { fail(::UMI - Read 1 Length must be greater than 0 if UMI - Read 1 Start From Cycle is greater than 0.::) }; if (step.hasValue(::UMI - Read 2 Length::) && !step.hasValue(::UMI - Read 2 Start From Cycle::)) { fail(::UMI - Read 2 Start From Cycle must be greater than 0 if UMI - Read 2 Length is greater than 0.::) }; if (!step.hasValue(::UMI - Read 2 Length::) && step.hasValue(::UMI - Read 2 Start From Cycle::)) { fail(::UMI - Read 2 Length must be greater than 0 if UMI - Read 2 Start From Cycle is greater than 0.::) }; if (!step.hasValue(::UMI - Read 1 Length::) && step.hasValue(::UMI - Read 2 Length::)) { fail(::UMI - Read 1 Length must be greater than 0 if UMI - Read 2 Length is greater than 0.::) }; if (step.hasValue(::Adapter Read 1::) && !step.::Adapter Read 1::.matches(::^[ACGT]+(\\+[ACGT]+){0,1}$::)) { fail(::Adapter Read 1 contains prohibited characters. Allowed characters are: ACGT+ and the adapter sequence cannot start or end with + or contain more than one +.::) }; if (step.hasValue(::Adapter Read 2::) && !step.::Adapter Read 2::.matches(::^[ACGT]+(\\+[ACGT]+){0,1}$::)) { fail(::Adapter Read 2 contains prohibited characters. Allowed characters are: ACGT+ and the adapter sequence cannot start or end with + or contain more than one +.::) };

Validation Script 2 Command Line

if ((step.::Aligner:: == ::BWA:: || step.::Aligner:: == ::TruSeq Amplicon Aligner::) && step.::Workflow:: != ::DNA Amplicon::) { fail(::Aligner field contains invalid value. Please refer to the \u005c\u0022Applicable analysis fields for the selected Workflow:\u005c\u0022 section for more information.::) }; if ((step.::Aligner:: == ::BWA-MEM:: || step.::Aligner:: == ::BWA-Backtrack Legacy::) && step.::Workflow:: == ::DNA Amplicon::) { fail(::Aligner field contains invalid value. Please refer to the \u005c\u0022Applicable analysis fields for the selected Workflow:\u005c\u0022 section for more information.::) }; if ((step.::Variant Caller:: == ::Starling:: || step.::Variant Caller:: == ::GATK::) && step.::Workflow:: == ::DNA Amplicon::) { fail(::Variant Caller field contains invalid value. Please refer to the \u005c\u0022Applicable analysis fields for the selected Workflow:\u005c\u0022 section for more information.::) }; if (step.::Variant Caller:: == ::Somatic:: && step.::Workflow:: != ::DNA Enrichment:: && step.::Workflow:: != ::DNA Amplicon::) { fail(::Variant Caller field contains invalid value. Please refer to the \u005c\u0022Applicable analysis fields for the selected Workflow:\u005c\u0022 section for more information.::) }; if (step.::Variant Caller:: == ::Germline:: && step.::Workflow:: != ::DNA Amplicon::) { fail(::Variant Caller field contains invalid value. Please refer to the \u005c\u0022Applicable analysis fields for the selected Workflow:\u005c\u0022 section for more information.::) }; step.::Indel-Realignment-Key:: = ::::; step.::Indel-Realignment-Value:: = ::::; step.::Aligner-Key:: = ::::; step.::Aligner-Value:: = ::::; step.::Variant-Caller-Value:: = ::::; if (step.::Workflow:: == ::DNA Amplicon::) { if (step.::Indel Realignment:: == ::Yes::) { step.::Indel-Realignment-Key:: = ::variantcallerrealignindels::; step.::Indel-Realignment-Value:: = ::1::; }; else if (step.::Indel Realignment:: == ::No::) { step.::Indel-Realignment-Key:: = ::variantcallerrealignindels::; step.::Indel-Realignment-Value:: = ::0::; }; if (step.::Aligner:: == ::TruSeq Amplicon Aligner::) { step.::Aligner-Key:: = ::aligner::; step.::Aligner-Value:: = ::Amplicon::; }; else if (step.::Aligner:: == ::BWA::) { step.::Aligner-Key:: = ::aligner::; step.::Aligner-Value:: = ::BWA::; }; }; if (step.::Workflow:: != ::DNA Amplicon::) { if (step.::Indel Realignment:: == ::Yes::) { step.::Indel-Realignment-Key:: = ::indelrealignment::; step.::Indel-Realignment-Value:: = ::GATK::; }; else if (step.::Indel Realignment:: == ::No::) { step.::Indel-Realignment-Key:: = ::indelrealignment::; step.::Indel-Realignment-Value:: = ::None::; }; if (step.::Aligner:: == ::BWA-Backtrack Legacy::) { step.::Aligner-Key:: = ::runbwaaln::; step.::Aligner-Value:: = ::1::; }; else if (step.::Aligner:: == ::BWA-MEM::) { step.::Aligner-Key:: = ::runbwaaln::; step.::Aligner-Value:: = ::0::; }; }; if (step.hasValue(::Variant Caller::)) { if (step.::Variant Caller:: == ::None::) { step.::Variant-Caller-Value:: = ::::; }; if (step.::Variant Caller:: == ::GATK::) { step.::Variant-Caller-Value:: = ::GATK::; }; else if (step.::Variant Caller:: == ::Starling::) { step.::Variant-Caller-Value:: = ::Starling::; }; else if (step.::Variant Caller:: == ::Germline::) { step.::Variant-Caller-Value:: = ::PiscesGermline::; }; else if (step.::Variant Caller:: == ::Somatic:: && step.::Workflow:: == ::DNA Amplicon::) { step.::Variant-Caller-Value:: = ::PiscesSomatic::; }; else if (step.::Variant Caller:: == ::Somatic:: && step.::Workflow:: != ::DNA Amplicon::) { step.::Variant-Caller-Value:: = ::Somatic::; }; }; if (step.hasValue(::Manifest::)) { step.::Manifest-Section:: = ::[Manifests]::; }; else { step.::Manifest-Section:: = ::::; }

Set Next Step - Advance Automation

This automation advances samples to the next step in the protocol. The automation is automatically triggered by exiting the Record Details screen.

The default command line is as follows.

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t false \
-h false \
-exp 'nextStep = ::ADVANCE::' \
-log {compoundOutputFileLuid3}"

Master Step Fields

Most fields configured on the Denature and Dilute (MiSeq v3.2) step display on the Record Details screen in the Step Data table.

These fields are manually populated at run time. The values are then used to generate the sample sheet.

Denature & Dilute (MiSeq v3.2) Master Step Field Configuration

Field Name	Field Type	Options	Additional Options and Drop-Down Items
Adapter Read 1	Text
Adapter Read 2	Text
Aligner	Text Dropdown		Presets None (default) BWA-MEM BWA-Backtrack Legacy BWA TruSeq Amplicon Aligner
Aligner-Key ℹ Used for sample sheet generation	Text	Read Only	Hidden
Aligner-Value ℹ Used for sample sheet generation	Text	Read Only
Annotation	Text Dropdown		Presets None (default) RefSeq Ensembl
Applicable analysis fields for the selected Workflow	Multiline Text	Read Only	ℹ Select a value from the drop-down list in the upper-right corner (below NEXT STEPS).
Comment	Multiline Text
Custom Primers	Text Dropdown		Presets None (Default) Read 1 Index Read 2 Read 1, Index Read 1, Index, Read 2 Read 1, Read 2 Index, Read 2
Description	Text
Experiment Name	Text	Required Field
Export to gVCF	Text Dropdown		Presets None (default) Yes No
Flag PCR Duplicates	Text Dropdown		Presets None (default) Yes No
Genome Folder	Text
Indel Realignment	Text Dropdown		Presets None (default) Yes No
Indel Repeat Filter Cutoff	Text Dropdown		Presets None (default) Yes No
Indel-Realignment-Key ℹ Used for sample sheet generation	Text	Read Only	Hidden
Indel-Realignment-Value ℹ Used for sample sheet generation	Text	Read Only	Hidden
Manifest	Text
Manifest Padding	Text Dropdown		Presets None (default) 0 50 100 150 200 250
Manifest-Section ℹ Used for sample sheet generation	Text	Read Only	Hidden
Picard HS Metric Reporting	Text Dropdown		Presets None (default) Yes No
Read 1 Cycles	Numeric Dropdown	Required Field Custom Entries	Presets 251 (Default) 151 101 76 51 Range = 1–1000 Decimal places displayed = 0
Read 2 Cycles	Numeric Dropdown	Required Field Custom Entries	Presets 251 (Default) 151 101 76 51 Range = 0–1000 Decimal places displayed = 0
Read Stitching	Text Dropdown		Presets None (default) Yes No
Reverse Complement	Text Dropdown		Presets None (default) Yes No
UMI - Read 1 Length	Numeric		Minimum value: 1
UMI - Read 1 Start From Cycle	Numeric		Minimum value: 1
UMI - Read 2 Length	Numeric		Minimum value: 1
UMI - Read 2 Start From Cycle	Numeric		Minimum value: 1
Validation Script 1 ℹ Used for automation	Multiline Text	Required Field Read Only	Hidden
Validation Script 2 ℹ Used for automation	Multiline Text	Required Field Read Only	Hidden
Variant Caller	Text Dropdown		Presets None (Default) GATK Germline Somatic Starling
Variant Caller Depth Filter	Numeric		Minimum value: 10 Maximum value: 10000
Variant Quality Filter	Numeric		Minimum value: 2 Maximum value: 1000
Variant Frequency Percentage	Numeric		Minimum value: 0.05 Maximum value: 100 Decimal places displayed = 2
Variant-Caller-Value ℹ Used for sample sheet generation	Text	Read Only	Hidden
Workflow	Text Dropdown	Required Field	Presets GenerateFASTQ LibraryQC Resequencing DNA Enrichment DNA Amplicon

Groups of Defaults

Resequencing

• Workflow = Resequencing

• Read 1 Cycles = 251

• Custom Primers = None

• Aligner = BWA-MEM

• Annotation = None

• Export to gVCF = No

• Flag PCR Duplicates = Yes

• Genome Folder = Required

• Indel Realignment = No

• Indel Repeat Filter Cutoff = None

• Manifest = None

• Manifest Padding = None

• Picard HS Metric Reporting = None

• Reverse Complement = None

• Read Stitching = None

• Variant Caller = GATK

• Applicable analysis fields for the selected Workflow =

  • Aligner (BWA-Backtrack Legacy, BWA-MEM)

  • Export to gVCF

  • Flag PCR Duplicates

  • Genome Folder (fill in the folder path)

  • Indel Realignment

  • Variant Caller (Starling, GATK)

Library QC

• Workflow = Library QC

• Read 1 Cycles = 251

• Custom Primers = None

• Aligner = None

• Annotation = None

• Export to gVCF = None

• Flag PCR Duplicates = Yes

• Genome Folder = Required

• Indel Realignment = None

• Indel Repeat Filter Cutoff = None

• Manifest = None

• Manifest Padding = None

• Picard HS Metric Reporting = None

• Reverse Complement = None

• Read Stitching = None

• Variant Caller = None

• Applicable analysis fields for the selected Workflow =

  • Flag PCR Duplicates

  • Genome Folder (fill in the folder path)

GenerateFastQ

• Workflow = GenerateFastQ

• Read 1 Cycles = 251

• Custom Primers = None

• Aligner = None

• Annotation = None

• Export to gVCF = None

• Flag PCR Duplicates = None

• Genome Folder = None

• Indel Realignment = None

• Indel Repeat Filter Cutoff = None

• Manifest = None

• Manifest Padding = None

• Picard HS Metric Reporting = None

• Reverse Complement = None

• Read Stitching = None

• Variant Caller = None

• Applicable analysis fields for the selected Workflow = None

DNA Enrichment

• Workflow = DNA Enrichment

• Read 1 Cycles = 251

• Custom Primers = None

• Aligner = BWA-MEM

• Annotation = None

• Export to gVCF = None

• Flag PCR Duplicates = Yes

• Genome Folder = Required

• Indel Realignment = Yes

• Indel Repeat Filter Cutoff = None

• Manifest = Required

• Manifest Padding = 150

• Picard HS Metric Reporting = No

• Reverse Complement = None

• Read Stitching = None

• Variant Caller = Starling

• Applicable analysis fields for the selected Workflow =

  • Aligner (BWA-Backtrack Legacy, BWA-MEM)

  • Flag PCR Duplicates

  • Genome Folder (fill in the folder path)

  • Indel Realignment

  • Indel Repeat Filter Cutoff (if Variant Caller is Somatic)

  • Manifest (fill in the file name)

  • Manifest Padding

  • Picard HS Metric Reporting

  • Variant Caller (Starling, Somatic, GATK)

  • Variant Frequency Percentage (if Variant Caller is Somatic)

DNA Amplicon

• Workflow = DNA Amplicon

• Read 1 Cycles = 251

• Custom Primers = None

• Aligner = BWA

• Annotation = RefSeq

• Export to gVCF = None

• Flag PCR Duplicates = None

• Genome Folder = Required

• Indel Realignment = Yes

• Indel Repeat Filter Cutoff = None

• Manifest = Required

• Manifest Padding = None

• Picard HS Metric Reporting = None

• Reverse Complement = None

• Read Stitching = None

• Variant Caller = Germline

• Variant Caller Depth Filter = 10

• Variant Quality Filter = 30

• Applicable analysis fields for the selected Workflow =

  • Aligner (BWA, TruSeq Amplicon Aligner)

  • Annotation (RefSeq, Ensembl)

  • Genome Folder (fill in the folder path)

  • Indel Realignment

  • Manifest (fill in the file name)

  • Read stitching (if Aligner is TruSeq Amplicon Aligner)

  • Variant Caller (Germline, Somatic)

  • Variant Caller Depth Filter (10–10000)

  • Variant Frequency Percentage (if Variant Caller is Somatic)

  • Variant Quality Filter (2–1000)

Global Fields

The following table shows the global fields that are configured to display on the Queue, Ice Bucket, and Record Details screens of the Denature and Dilute (MiSeq v3.2) step:

The Submitted Sample field, Progress, (added from previous MiSeq workflow) is obsolete in MiSeq v3.2.

Global Field Configuration (Submitted Sample)

Field Name	Field Type	Field Constraints/Options	Preset Values/Additional Options and Drop-Down Items
Read Length ℹ Displays on Queue & Ice Bucket screens	Text
Sequencing Method ℹ Displays on Queue & Ice Bucket screens	Text Dropdown	Custom Entries	Presets Single Read Paired End Read Indexed Single Read Indexed Paired End Read

Global Field Configuration (Derived Sample)

Field Name	Field Type	Field Constraints/Options	Preset Values/Additional Options and Drop-Down Items
Final Loading Concentration ℹ Displays on Record Details screen	Numeric Dropdown	Required Field Custom Entries	Presets 225 400 Decimal places displayed = 0

Step File Placeholders

Placeholders for the following files are configured on the Record Details screen of the Denature and Dilute (MiSeq v3.2) step.

Lab Tracking Form

• Manually uploaded

• This item in Clarity LIMS allows the lab scientist to attach a lab-specific tracking form to the step manually.

MiSeq SampleSheet

• Automatically attached

• This CSV file is automatically generated by Clarity LIMS for use with the MiSeq system. The file can be opened as a text file or an Excel spreadsheet.

MiSeq SampleSheet Generation Log

• Automatically attached

• This log file is automatically generated by Clarity LIMS. The log file captures any errors that Clarity LIMS might encounter when generating the sample sheet.

Log File

• Automatically attached

• This log file is automatically generated by Clarity LIMS. The log file captures the status of the EvaluateDynamicExpression script that is invoked by the Set Next Step - Advance automation.

Step 3: MiSeq Run (MiSeq v3.2)

Set Next Step - Advance Automation

This automation advances samples to the next step in the protocol. The automation is automatically triggered by exiting the Record Details screen.

The default command line is as follows.

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t false \
-h false \
-exp 'nextStep = ::ADVANCE::' \
-log {compoundOutputFileLuid5}"

Master Step Fields

The following fields are configured on the MiSeq Run (MiSeq v3.2) step. These fields display on the Record Details screen at run time. The read-only fields are automatically populated at the end of the run.

MiSeq Run (MiSeq v3.2) Master Step Field Configuration

Field Name	Field Type	Options	Additional Options and Drop-Down Items
Chemistry	Text	Read Only
Comment	Multiline Text
Experiment Name	Text	Read Only
Finish Date	Date	Read Only
Flow Cell ID	Text	Read Only
Flow Cell Version	Text	Read Only
Index 1 Read Cycles	Numeric	Read Only	Decimal Places Displayed: 0
Index 2 Read Cycles	Numeric	Read Only	Decimal Places Displayed: 0
Output Folder	Text	Read Only
PR2 Bottle ID	Text	Read Only
Read 1 Cycles	Numeric	Read Only	Decimal Places Displayed: 0
Read 2 Cycles	Numeric	Read Only	Decimal Places Displayed: 0
Reagent Cartridge ID	Text	Read Only
Reagent Cartridge Part #	Text	Read Only
Run ID	Text	Read Only
Run Type	Text	Read Only
Status	Text	Read Only
Workflow	Text	Read Only

Global Fields

There are several sample and measurement global fields that are displayed on the Record Details screen of the MiSeq Run (MiSeq v3.2) step. These fields are autopopulated at the end of the sequencing run.

For more information, refer to Sequencing Results Parsing.

Step File Placeholders

Placeholders for the following files are configured on the Record Details screen of the MiSeq Run (MiSeq v3.2) step:

• Illumina Run Report (automatically attached)

• Link to Run Folder (automatically attached)

• Run Parameters (automatically attached)

• Run Info (automatically attached)

• Lab Tracking Form (manually uploaded)

• Log File (automatically attached)

For details, refer to Generated and Captured Files.

Sample Sheet Generation

Sample sheet generation occurs in the Denature & Dilute (MiSeq v3.2) step. This step places samples on the container loaded in the system.

The default configuration provides only the Validate Run Setup and Generate MiSeq SampleSheet automation. This automation uses the Template File Generator (DriverFileGenerator.jar) and a template file to generate a CSV format file for use with the MiSeq Control Software (MCS).

The sample sheet content is determined by the fields on the Record Details screen of the step in the Step Data table. The values entered into these fields are used to populate the sample sheet.

To customize the template used to create the sample sheet, you can insert additional columns.

The following additional details are available:

• For a sample template that you can download and customize for the lab, refer to Illumina Instrument Sample Sheets (NGS v5.17 & later).
• For instructions on customizing the template, refer to Creating Template Files.

Sequencing Results Parsing

The MiSeq Run (MiSeq v3.2) step records information for the flow cell lane and generates a report summarizing the results. In addition, run parameters, run info, and a link to the run folder are automatically captured.

Generated and Captured Files

The following table describes the run information files, reports, placeholders, and links that Clarity LIMS automatically generates or captures during a sequencing run:

Run Information Generated or Captured by MiSeq Run (MiSeq v3.2) Step

Item	Description
Run Info Run Parameters	These XML files are automatically captured by Clarity LIMS from the run folder of the system. They include the key run parameters that are parsed out into step custom fields in Clarity LIMS.
Link to Run Folder	This link is the path to the network run folder where the data that was captured from the system during the run is stored. The link is automatically generated by Clarity LIMS.
Illumina Run Report	This report provides key information about the run and the samples on the flow cell. The report is automatically generated by Clarity LIMS. Information includes the flow cell ID, run directory location, and primary analysis metrics for the sequencing run. Information is summarized per flow cell lane for the entire run and individual reads for paired-end runs. These metrics are compared against the per lane averages of the sequencing run. The per lane averages are calculated using metrics from the last five sequencing runs. Any values outside of one standard deviation are highlighted.
Lab Tracking Form	This item in Clarity LIMS allows you to manually attach a lab-specific tracking form to the step.

Metadata

The following list shows metadata that Clarity LIMS automatically captures from the Illumina sequencing software as part of a sequencing run. This information is gathered from various run result files and events.

• Chemistry

• Experiment Name (entered in software)

• Finish Date (run completion date)

  • If the End Run event contains a date in the format YYYY-MM-DD, Finish Date is set to the date in the event file.

  • If the End Run event does not contain a date or the date is in the wrong format, Finish Date is set to the date when the event file is processed.

• Flow Cell ID

• Flow Cell Version

• Index 1 Read Cycles (intended Index cycles)

• Index 2 Read Cycles (intended Index cycles)

• Output Folder (run folder root)

• PR2 Bottle ID

• Reagent Cartridge ID

• Reagent Cartridge Part #

• Read 1 Cycles

• Read 2 Cycles

• Run ID (the unique run ID)

• Run Type

• Status (current status of the sequencing run on the instrument)

• Workflow

Primary Analysis Metrics

The following table lists the real-time analysis (RTA) primary analysis metrics Clarity LIMS automatically captures and records per read, for samples in each flow cell lane. These metrics are captured upon run completion and are stored as fields in the Sample Details table on the Record Details screen.

To see both per read and per lane metrics, expand the output.

RTA Primary Analysis Metrics Captured by MiSeq Run (MiSeq v3.2) Step

Per Read LIMS Field Name (Stored on derived sample/analyte input to the step)	Per Lane LIMS Field Name (Stored in measurement placeholders in the Sample Details table on the Record Details screen)
% Aligned R1	% Aligned R1
% Aligned R2	% Aligned R2
% Bases >=Q30 R1	% Bases >=Q30 R1
% Bases >=Q30 R2	% Bases >=Q30 R2
% Error Rate R1	% Error Rate R1
% Error Rate R2	% Error Rate R2
	% Phasing R1
	% Phasing R2
	% Prephasing R1
	% Prephasing R2
	%PF R1
	%PF R2
	Cluster Density (K/mm^2) R1
	Cluster Density (K/mm^2) R2
Intensity Cycle 1 R1	Intensity Cycle 1 R1
Intensity Cycle 1 R2	Intensity Cycle 1 R2
	Reads PF (M) R1
	Reads PF (M) R2
Yield PF (Gb) R1	Yield PF (Gb) R1
Yield PF (Gb) R2	Yield PF (Gb) R2

How It Works

1. The sequencing service runs on the Clarity LIMS server. The service detects event files that instrument RTA produces as the run progresses. The event files let the service know where to find the run data.

2. As the run data are written out and the End Run event is detected, the following events occur:

   • The data are matched to the step based on the reagent cartridge ID that was entered or scanned on the Denature and Dilute (MiSeq v3.2) step.

   • Read-only field values on the Record Details screen are populated accordingly.

3. When the service has finished processing the end run event and updating the fields in Clarity LIMS, the sequencing service generates the report and attaches it to the step.

Scripts and Files Installed

This integration requires components installed with the Illumina Preset Protocols (IPP).

The Illumina MiSeq Integration Package v8.3.0 RPM installs the scripts and files listed in the following table.

Component	Location	Description
configure_extensions_miseq_sequencingservice-v8.sh	/opt/gls/clarity/config/	Script that installs the service properties in the database.
log4j.xml	/opt/gls/clarity/extensions/Illumina_MiSeq/v8/SequencingService/conf	File containing the settings for logging the sequencing jar.
miseq-sequencing-v8.jar	/opt/gls/clarity/extensions/Illumina_MiSeq/v8/SequencingService	Jar file containing API-based Clarity LIMS extensions used for capturing run results and report generation.
InterOp libraries	/opt/gls/clarity/extensions/Illumina_MiSeq/v8/lib	Illumina shared library for parsing InterOp data files.
MiSeqSamplesheetv3.csv ℹ Installed by IPP	/opt/gls/clarity/extensions/conf/driverfiletemplates	Template file used for file generation.

Properties Installed

Refer to Integration Properties Details for the properties installed with Illumina MiSeq Integration v8.3.0.

Consumables Installed

Reagent Categories and Kits

Reagent categories or label groups are installed with the IPP workflow configuration slices.

The MiSeq Reagent Kit is included in the Illumina MiSeq Integration.

Control Types

The PhiX v3 control type is included in the Illumina MiSeq Integration.

Container Types

The following container types are included in the Illumina MiSeq Integration:

• MiSeq Reagent Cartridge

• 96 well plate

• Tube

All one-dimensional container types with both numeric rows and numeric columns are supported.

Instrument Integration

To make sure that the Illumina instrument warranty remains valid, the instrument integration must be performed and maintained by the Clarity LIMS Support team. To perform this integration, the Support team requires remote access to the system while it is idle.

The following steps are performed by the Clarity LIMS Support team when configuring the sequencing for use with the Illumina MiSeq Integration.

1. Create a directory on the local computer to hold the batch files. These batch files write event files to the network attached storage (NAS) shares.

2. Create a directory on the NAS to hold the event files.

3. Modify Illumina software configuration files to call the batch files that create the event files.

4. Update sequencing service default properties to match the specifics of the installation.

Rules and Constraints

The Illumina MiSeq Integration operates with the following constraints:

• The reagent cartridge ID must be unique. There should not be multiple reagent cartridge containers in the system with the same name.

• The reagent cartridge ID must be scanned as the reagent cartridge Container Name on the Denature & Dilute (MiSeq v3.2) step.