MiSeqDx Sample Sheet Generation (v1.11.0 and later)

Package Version: BaseSpace Clarity LIMS MiSeqDx (v1.11.0 and later)

Overview

The Illumina MiSeqDx Integration Package allows for automatic generation of a sample sheet to be used with the MiSeqDx instrument software. The format of this sample sheet is designed for the instrument when it is running in Diagnostic mode.

⚠️ Note

• If you are running the MiSeqDx instrument in Research Use Only (RUO) mode, see Sample Sheet Generation section in the Configuration guide for MiSeq version-of-interest.

• MiSeqDx does not support bcl2fastqv2 sample sheet generation.

How sample sheet generation works

Sample sheet generation is configured on the step prior to the sequencing run – Denature, Dilute and Load Sample, which is the step where samples are placed on the flow cells or reagent cartridges that will be placed in the instrument.

The sample sheet is generated by means of a script, which the lab user initiates by clicking a button on the Record Details screen of the step. This generates a sample sheet file for the container loaded during the step, where the name of the sample sheet will be

<reagent cartridge barcode ID>.csv

The user then downloads the sample sheet from the LIMS and uploads it to the instrument software.

MiSeqDx Assays Support

The following assays are supported by the MiSeqDx sample sheet generation script:

• CF 139-Variant Assay

• CF Clinical Sequencing Assay

Configured Master Step Fields/Step UDFs

Sample sheet format is controlled via master step field / step user defined field (UDF) configuration, where key step fields are pre-populated with values specific to each protocol step. These values are not editable, and their configuration should not be modified.

For details, see Sample Sheet Data and File Format and Contents sections below.

The fields listed in the following table are available on the Denature, Dilute and Load Sample step and will be placed into the sample sheet.

Field Name	Field Type	Required?	Notes
Experiment Name	Text	No	Entered by the user
Workflow	Text	Yes	Value set to Amplicon for CF 139-Variant Assay and CF Clinical Sequencing Assay
Description	Text	No	Entered by the user
Assay	Text	No	Configured with the following preset values CF 139-Variant CF Clinical Sequencing /ul>
Amplicon Workflow Type	Text	No	Configured with the following preset values CF139VARIANTASSAY CFCLINICALSEQUENCINGASSAY
Application	Text	Yes	Configured with the following preset values CF 139-Variant Assay CF Clinical Sequencing Assay
PhiX Control added?	Check box	No	Default set to false
VariantCaller	Text	No	Value set to Starling, a legacy variant caller
Variant Min Quality Cutoff	Numeric	No	Value set to 100
GenomeFolder	Text	Yes	Required for secondary analysis
Control CAT Manifest	Text	No	Preset value CFTRManifest.txt
Custom CAT Manifest	Text	No	Manifest file for custom assay
Read 1 Cycles	Numeric	Yes	Configured with range 0-1000. Value set to 151
Read 2 Cycles	Numeric	Yes	Configured with range 0-1000. Value set to 151

Submitted Sample Global Fields/UDFs

Field Name	Field Type	Required?	Notes
Reference Genome	Text	No	Optionally used to populate the GenomeFolder value for individual samples in the sample sheet.
Control?	Text	No	Used to indicate a control sample that is represented as a submitted sample in the LIMS.

Script Parameters and Usage

Parameter	Description	Required?	Notes
u, username	LIMS username	Yes
p, password	LIMS password	Yes
i, processURI	LIMS process URI	Yes
c, csvFileLimsIds	Sample sheet CSV file LIMS ID	Yes	May be provided multiple times
e, errorLogFileName	Log file name	Yes
l, useProjectLimsID	Project LIMS ID will be used instead of project name in the Project column of the sample sheet	No	Accepted values: true or false. Provide with quotes e.g. -l 'true'
s, useSampleLimsID	Should be set to true Sample LIMS ID will be used instead of sample name in the SampleName column of the sample sheet	No	Accepted values: true or false. Provide with quotes e.g. -s 'true' See Enabling unique FASTQ file names in Configuration Options
a, appendLimsID	Should be set to false LIMS ID of the protocol step will be appended to sample names in the SampleName column of the sample sheet.	No	Accepted values: true or false. Provide with quotes e.g. -a 'true'

Usage

Below is the default command line that ships with the Denature, Dilute and Load Sample (CF 139-Variant Assay) step.

bash -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -u {username} -p {password} -i {processURI:v2} script:setUDF -f 'Progress' -t '//input/@uri->//sample/@uri' -v 'Library ready for sequencing' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/miseqdx/miseqdx-extensions.jar -u {username} -p {password} -i {processURI:v2} script:generate_miseqdx_sample_sheet -c {compoundOutputFileLuid1} -e {compoundOutputFileLuid2} -useSampleLimsID true && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -u {username} -p {password} -i {processURI:v2} script:labelNonLabeledOutputs -l 'NoIndex'"

Support for Container Types

Single well container types, and all one-dimensional container types with both numeric rows and numeric columns, are supported.

Sample Sheet Data

The following table lists and describes the fields included in the MiSeqDx sample sheet.

Note the following:

• If no upstream pooling is detected, LIMS will populate the sample sheet with the SampleID and SampleName of the submitted sample. Other fields are populated with data from the samples that were input to the step (i.e. derived samples).

• If upstream pooling is detected, LIMS will populate the sample sheet with the first upstream pooled inputs found – not with the submitted sample or step input fields.

Control samples may be one of the following:

• Built-in BaseSpace Clarity LIMS control samples

• Submitted samples with field / UDF Control? set to true

Header Section

Field Name	Description	Required?	Notes
WMFileVersion	Illumina Worklist Manager Version number.	No
Date	The date the sample sheet was generated.	No
Workflow	Master step field/Step UDF of the same name.	Yes
Application	Populated with CF 139-Variant Assay or CF Clinical Sequencing Assay.	Yes
Assay	Master step field/Step UDF of the same name.	No
Description	Master step field/Step UDF of the same name.	No
Chemistry	The recipe fragments used to build the run-specific recipe.	Yes	Populated with Amplicon

Manifests Section

ℹ️ If a value is provided for only a single CAT manifest file, then all samples in the sample sheet will be given the designation (A) associated with that CAT type.

Field Name	Description	Required?	Notes
A	Master step field/Step UDF Control CAT Manifest. Typically a path to a file.	No	Must be a real path. Convention indicates this is the Control CAT. Control samples will be given the designation A in the Data section.
B	Master step field/Step UDF Control CAT Manifest. Typically a path to a file.	No	Must be a real path. Convention indicates this is the Control CAT. Control samples will be given the designation B in the Data section.

Reads Section

Field Name	Description	Required?	Notes
ℹ️ Read cycle entry is listed beneath the [Reads] heading, in the first column of the spreadsheet.	Master step field/Step UDF Read 1 Cycles	Yes	Index reads are determined by the MOS, based on the indexes on the inputs.
ℹ️ Read cycle entry is listed beneath the [Reads] heading, in the first column of the spreadsheet.	Master step field/Step UDF Read 2 Cycles	No	Index reads are determined by the MOS, based on the indexes on the inputs.

ℹ️ For single read, one entry is listed beneath the [Reads] heading, in the first column of the spreadsheet. For paired end, two entries are listed.

Settings Section

Field Name	Description	Required?	Notes
AmpliconWorkflowType	Master step field/Step UDF Amplicon Workflow Type.	No	Populated with values CF139VARIANTASSAY or CFCLINICALSEQUENCINGASSAY
VariantCaller	Master step field/Step UDF of the same name.	No
VariantMinimumQualCutoff	Master step field/Step UDF Variant Min Quality Cutoff.	Yes

Data Section

Field Name	Description	Required?	Notes
Sample_ID	Populated with the LIMS ID of the sample if pooled, or the LIMS ID of the submitted sample if not pooled.	Yes
Sample_Name	Populated with the sample name if pooled, or the submitted sample name if not pooled.	Always present	If script parameter useSampleLimsID is provided on the command line, the LIMS ID of the sample will be used instead of the name. The additional -a command line option appends the LIMS ID to the end of this value, e.g. Sample1-1234 See Script Parameters and Usage.
Sample_Plate	Name of the Container that the Sample resides in as recorded in the LIMS.	Always present
Sample_Well	Container well location of the sample. If a sample is part of a pool, this will list the well location of the sample that was added to the pool.	Always present
Sample_Project	The name of the project in the LIMS, that the sample belongs to.	Always present
Control	Blank for a normal sample. Populated with value positive when Positive Control for MiSeqDx has been added to the pool. Populated with value negative when Negative Control for MiSeqDx has been added to the pool.	No	Controls must have an index. Controls in these cases look just like the other samples.
index	Determined from the reagent label. Uses the Sequence attribute value from Index Reagents. Dual index reagents will contain a hyphen-separated DNA sequence; this field will use the first half of that value.	Yes, if more than 1 input
I7_Index_ID	Determined from the name of the index reagent type. Dual index names will be hyphen-separated; this field will use the first half of that value.	Yes, if more than one input.
index2	Determined from the reagent label. Uses the Sequence attribute value from Index Reagents. Dual index reagents will contain a hyphen-separated DNA sequence; this field will use the second half of that value.	No
I5_Index_ID	Determined from the name of the index reagent type. Dual index names will be hyphen-separated; this field will use the second half of that value.	No
Manifest	A	Yes	Value determined by the entries in the Manifests Section.
GenomeFolder	If master step field/step UDF Use submitted sample details for Genome Folder location is true, this is populated with the value of submitted sample global field/UDF Reference Genome. Otherwise, populated with the value of master step/step UDF GenomeFolder.	Yes, if Use submitted sample details for Genome Folder location is true	Folder path for ReferenceGenomes used for secondary analysis.
Description		No

File Format and Contents

This section outlines the format and contents of the generated sample sheet and associated log file.

When validating the installation of your integration, refer to this information to ensure that the sample sheet and log files are correctly generated.

MiSeqDx sample sheet

• The file is a comma-separated file.

• The file contains the following sections:

  • Header

  • Manifests

  • Reads

  • Settings

  • Data

• The file is populated with data from the samples in the step. If pooled, each sample in the pool is represented as a separate, demultiplexed entry.

• The entries are sorted by SampleWell and by SampleID.

• The data section of the file contains 11 columns.

MiSeqDx sample sheet log file

• The file is in HTML format.

• The file contains logging information and a success message if sample sheet generated successfully.

Configuration Options

Enabling unique FASTQ file names

To enable unique FASTQ file names per sequencing run, the EPP command on the process type must be configured to use the following parameter options:

• -useSampleLimsID – ensures unique entries in the SampleName column by using the sample LIMS ID instead of its name

• -appendLimsID – ensures unique names per run by appending the LIMS ID of the current step

For more information, see Script Parameters and Usage.

Rules and Constraints

• The step on which this script runs must be the step in which samples are placed on the flow cell(s) or reagent cartridge(s).

• The contents of the sample sheet are ordered by SampleWell and then ordered by SampleID.

• Project and sample names in the sample sheet cannot contain illegal characters. Characters not allowed are the space character and the following: ? ( ) [ ] / \ = + < > : ; " ' , * ^ | &

• Illegal characters will be replaced with an underscore "_"

• The destination container type (flow cell or reagent cartridge) must be must be either single well or a one-dimensional container type with both numeric rows and numeric columns. Back to top