Illumina MiSeqDx 1.x Sample Sheet Generation
Package Version: BaseSpace Clarity LIMS MiSeqDx
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.
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Note:
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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.
The following assays are supported by the MiSeqDx sample sheet generation script:
| • | CF 139-Variant Assay |
| • | CF Clinical Sequencing Assay |
| • | Universal Kit |
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.
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Field Name |
Field Type |
Required? |
Notes |
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Experiment Name |
Text |
No |
Entered by the user |
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Workflow |
Text |
Yes |
Value set to Amplicon for CF 139-Variant Assay and CF Clinical Sequencing Assay Value set to Custom Amplicon for Universal Kit |
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Description |
Text |
No |
Entered by the user |
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Assay |
Text |
No |
Configured with the following preset values:
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Amplicon Workflow Type |
Text |
No |
Configured with the following preset values:
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Application |
Text |
Yes |
Configured with the following preset values:
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PhiX Control added? |
Check box |
No |
Default set to false |
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VariantCaller |
Text |
No |
Value set to Starling, a legacy variant caller |
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Variant Min Quality Cutoff |
Numeric |
No |
Value set to 100 |
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GenomeFolder |
Text |
Yes |
Required for secondary analysis |
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Control CAT Manifest |
Text |
No |
Preset value CFTRManifest.txt |
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Custom CAT Manifest |
Text |
No |
Manifest file for custom assay |
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Read 1 Cycles |
Numeric |
Yes |
Configured with range 0-1000. Value set to 151 |
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Read 2 Cycles |
Numeric |
Yes |
Configured with range 0-1000. Value set to 151 |
Submitted sample global fields/UDFs
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Field Name |
Field Type |
Required? |
Notes |
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Reference Genome |
Text |
No |
Optionally used to populate the GenomeFolder value for individual samples in the sample sheet. |
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Control? |
Text |
No |
Used to indicate a control sample that is represented as a submitted sample in the LIMS. |
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Parameter |
Description |
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u, username |
LIMS username (Required) |
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p, password |
LIMS password (Required) |
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i, processURI |
LIMS process URI (Required) (lowercase I) |
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c, csvFileLimsIds |
Sample sheet CSV file LIMS ID (Required - may be provided multiple times) |
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e, errorLogFileName |
Log file name (Required) |
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l, useProjectLimsID |
Project LIMS ID will be used instead of project name in the Project column of the sample sheet (Optional) Accepted values: true or false. Provide with quotes e.g. -l 'true' (lower case L) |
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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 (Optional) (See Enabling unique FASTQ file names) Accepted values: true or false. Provide with quotes e.g. -s 'true' |
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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. (Optional) 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) 1.0 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'"
Single well container types, and all one-dimensional container types with both numeric rows and numeric columns, are supported.
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. |
About controls
Control samples may be one of the following:
| • | Built-in BaseSpace Clarity LIMS control samples |
| • | Submitted samples with field / UDF Control? set to true |
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Field Name |
Contents |
Comments |
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[Header] |
Optional |
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WMFileVersion |
Illumina Worklist Manager Version number |
Optional |
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Date |
The date the sample sheet was generated. |
Optional |
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Workflow |
Master step field / Step UDF of the same name. |
Required |
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Application |
Populated with CF 139-Variant Assay or Cystic Fibrosis 139 Variant Assay or MiSeqDx Universal. |
Required |
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Assay |
Master step field / Step UDF of the same name. |
Optional |
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Description |
Master step field / Step UDF of the same name. |
Optional |
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Chemistry |
Populated with Amplicon. |
Required for all workflows. |
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[Manifests] |
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. |
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A |
Master step field / Step UDF Control CAT Manifest. Typically a path to a file. |
Optional, but must be a real path. Convention indicates this is the Control CAT. Control samples will be given the designation A in the Data section. |
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[Reads] |
Master step fields / Step UDFs of the same name Read 1 Cycles and Read 2 Cycles. |
Required. Read cycle entries are listed beneath the [Reads] heading, in the first column of the spreadsheet. One entry for single read, two entries for paired end. Index reads are determined by the MOS, based on the indexes on the inputs. Read 1 cycles is required for all workflows. Read 2 cycles is optional. |
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[Settings] |
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AmpliconWorkflowType |
Master step field / Step UDF Amplicon Workflow Type. Populated with values CF139VARIANTASSAY or CFCLINICALSEQUENCINGASSAY or MiSeqDx Universal |
Optional |
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VariantCaller |
Master step field / Step UDF of the same name. |
Optional |
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VariantMinimumQualCutoff |
Master step field / Step UDF Variant Min Quality Cutoff. |
Optional |
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[Data] |
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Sample_ID |
Populated with the LIMS ID of the sample if pooled, or the LIMS ID of the submitted sample if not pooled. |
Required for all workflows. |
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Sample_Name |
Populated with the sample name if pooled, or the submitted sample name if not pooled. 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 |
Always present, even if not populated. See Script parameters. |
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Sample_Plate |
Name of the Container that the Sample resides in as recorded in the LIMS. |
Always present. |
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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. |
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Sample_Project |
The name of the project in the LIMS, that the sample belongs to. |
Always present. |
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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. Controls must have an index.Controls in these cases otherwise look just like the other samples. |
Optional |
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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. |
Required if more than one input. |
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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. |
Required if more than one input. |
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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. |
Optional |
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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. |
Optional |
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Manifest |
A |
Required. Value determined by the entries in the Manifests section. |
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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. |
Required folder path for ReferenceGenomes used for secondary analysis. |
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Description |
Optional |
