NovaSeq X Settings
Standalone Sections
Header Section
Custom_*
No
Custom field used to capture run metadata.
String with ASCII characters except for * and the control characters CR and LF.
FileFormatVersion
Yes
Used to identify the sample sheet as a v2 sample sheet. This field must always exist in the header section with a value of 2.
Must always be 2.
InstrumentPlatform
No
Identifies the instrument platform to be used for the run.
For example, enter NovaSeqXSeries for NovaSeq X Series.
String with ASCII characters except for * and the control characters CR and LF.
InstrumentType
No
Identifies the instrument to be used for the run.
For example: if using NovaSeq X, populate the field with NovaSeq X.
String with ASCII characters except for * and the control characters CR and LF.
RunDescription
No
The run description can contain 255 alphanumeric characters, spaces, dashes, and underscores.
String with ASCII characters except for * and the control characters CR and LF.
RunName
Yes
The run name can contain 255 alphanumeric characters, spaces, dashes, and underscores.
String with ASCII characters except for * and the control characters CR and LF.
Reads Sections
Index1Cycles
No¹
Number of cycles in Index Read 1. Required if more than one sample is present in sample sheet.
Must be an integer ≥ 0.
Depending on your sequencing system and reagent kit, there can be limitations on the number of cycles in the Index Read.
Warning if values in range [1–5] inclusive.
If there is more than 1 sample per lane, must be > 0.
If OverrideCycles is present in the BCL_Settings section, must be consistent with the sum of the Index1 section of OverrideCycles.
Index2Cycles
No¹
Number of cycles in Index Read 2. Required if using dual indexes for demultiplexing.
Must be an integer ≥ 0.
Depending on your sequencing system and reagent kit, there can be limitations on the number of cycles in the Index Read.
Warning if values in range [1–5] inclusive.
If OverrideCycles is present in the BCL_Settings section, must be consistent with the sum of the Index1 section of OverrideCycles.
Read1Cycles
Yes
Number of cycles for Read 1.
Must be an integer > 0.
Warning if less than 26.
If OverrideCycles is present in the BCL_Settings section, must be consistent with the sum of the Read1 section of OverrideCycles.
Read2Cycles
Yes
Number of cycles for Read 2. Required only when running a paired-end sequencing run.
Must be an integer ≥ 0.
Warning if values in range [1–25] inclusive.
If OverrideCycles is present in the BCL_Settings section, must be consistent with the sum of the Read2 section of OverrideCycles.
¹ Depending on the run or library prep kit, this parameter might be required.
Sequencing Section
CustomIndex1Primer
No¹
Indicates if a Custom Index 1 primer is used for the run.
Values true and false allowed. Value true only allowed if Index1Cycles is specified.
CustomIndex2Primer
No¹
Indicates if a Custom Index 2 primer is used for the run.
Values true and false allowed. Value true only allowed if Index2Cycles is specified.
CustomRead1Primer
No¹
Indicates if a Custom Read 1 primer is used for the run.
Values true and false allowed. Value true only allowed if Read1Cycles is specified.
CustomRead2Primer
No¹
Indicates if a Custom Read 2 primer is used for the run.
Values true and false allowed. Value true only allowed if Read2Cycles is specified.
LibraryPrepKits
Yes
Identifies the library prep kit used for the run.
String with ASCII characters except for * and the control characters
CRandLF.If more than one library prep kit is being used, use semicolons to separate the names of the different library prep kits.
¹ Depending on the run or library prep kit, this parameter might be required.
Application Sections
For NovaSeq X Series, there is a limit of four application sections for onboard analysis or eight application sections for cloud analysis.
BCL Convert
BCLConvert_Settings
SoftwareVersion
Yes
The version of the software to be used to perform BCL conversion on the Sample_ID's that only exist in the BCL Convert Data section of the sample sheet. The version is specified using all three integers included in the DRAGEN version name. For example, 1.0.0.
FastqCompressionFormat
Yes
The compression format for the FASTQ output files. Allowed values are gzip or dragen.
GenerateFastqcMetrics
No
Enable/disable generation of FAST QC Metrics. Default = False. If included in the Sample Sheet, then all DRAGEN versions (for BCL Convert and non-BCL Convert workflows) must be 4.3.13 or later. Not applicable for the Cloud pipeline mode.
CreateFastqForIndexReads
No
TrimUMI
No
AdapterBehavior
No
AdapterStringency
No
MinimumTrimmedReadLength
No
MinimumAdapterOverlap
No
MaskShortReads
No
NoLaneSplitting
No
FindAdaptersWithIndels
No
IndependentIndexCollisionCheck
No
BCLConvert_Data
AdapterRead1
No
The sequence of the Read 1 adapter to be masked or trimmed. To trim multiple adapters, separate the sequences with a plus sign (+) indicating independent adapters that must be independently assessed for masking or trimming for each read. Characters must be A, C, G, or T.
A value of na (case insensitive) must be used if:
the AdapterRead1 field is placed in the Data section of the Sample Sheet, and
no adapter trimming is desired for the sample
AdapterRead2
No
See description of AdapterRead1, applied to AdapterRead2.
BarcodeMismatchesIndex1
No
Specifies barcode mismatch tolerance for Index 1. Possible values are 0, 1, or 2, or na. The default value is 1. Only allowed if Index is specified in RunInfo.xml file and in the Reads section of the Sample Sheet.
A value of na must be used if:
BarcodeMismatchesIndex1 exists in the Data section of the Sample Sheet, and
OverrideCycles exist in the data section, and
Index 1 is masked out for the sample in the OverrideCycles
BarcodeMismatchesIndex2
No
See description of BarcodeMismatchesIndex1, applied to BarcodeMismatchesIndex2.
OverrideCycles
No
Specifies the sequencing and indexing cycles to be used when processing the sequencing data. Must adhere to the Specifies the sequencing and indexing cycles to be used when processing the sequencing data. Must adhere to the following requirements: • Must be same number of fields (delimited by semicolon) as sequencing and indexing reads specified in RunInfo.xml and in the Reads section of the Sample Sheet. • Indexing reads are specified with I, sequencing reads are specified with Y, UMI cycles are specified with U, and trimmed reads are specified with N. • The number of cycles specified for each read must equal the number of cycles specified for that read in the RunInfo.xml file and in the Reads section of the Sample Sheet. • Only one Y or I sequence can be specified per read.
'I' cycles can only be specified for index reads
'Y' cycles can only be specified for genomic reads
The total number of cycles used for demultiplexing each index cannot exceed 27. Note that this includes all cycles between the first "I" cycle used by any sample and the last "I" cycle used by any sample within each index.
"I5N3;I5N3" : counts as 5 bases toward the limit of 27 for Index1, and as 5 bases toward the limit of 27 for Index2
"I4N1I3;I5N3": counts as 8 bases toward the limit of 27 for Index1, and as 5 bases toward the limit of 27 for Index2
The following are examples of OverrideCycles input: U8Y143;I8;I8;U8Y143 N10Y66;I6;N10Y66
For a sample sheet containing two samples having the following OverrideCycles:
Y151; I8N2; N10; Y151
Y151; N2I8; I8N2; Y151
the number of cycles used for demultiplexing sums to 18.
For sequencers with RunInfo.xml having "IsReverseComplement" set to "Y" for Index2, then Index2 is processed in reverse complement orientation. Any trimming should be applied to the end of the sequence as it transitions to the adapter, which means the "N" cycles specified in OverrideCycles must be in the beginning of the index read.
Example of 8bp i5 index with the last two (adapter) bases to be trimmed:
Forward i5: XXATCGCGGT
ReverseComp i5: ACCGCGATXX (this is the direction of sequencing)
OverrideCycles for Index2: N2I8
where XX is part of the adapter sequence.
Although Index2 is processed on the sequencer in reverse complement format, Index2 and OverrideCycles are entered in the Sample Sheet in forward, non-complemented format for user convenience (see diagram below).
Note that NovaSeq X has a RunInfo.xml with "IsReverseComplement" set to "Y" for Index2.
Sample_ID
Yes
ID for the sample with the following requirements: • Must be alphanumeric string with _ or - and no spaces. • Case sensitive (i.e., a Sample Sheet with the samples MySample and mysample is not allowed)
The same Sample_ID may exist on more than one row of the Sample Sheet (e.g., one sample spanning more than one lane)
Undetermined is not allowed as a Sample_ID
Lane
Yes
Specifies FASTQ files only for the samples with the specified lane number.
Must adhere to the following requirements:
Must be an integer
Value must be in the range of lanes specified in RunInfo.xml
Ranges are not supported with '-' or '+'
If not supplied, it is assumed that all samples are present in all lanes specified in the RunInfo.xml
If supplied, only lanes specified in the column will be converted from BCL to FASTQ
For NovaSeqX, values must be in the range of lanes specified in RunInfo.xml
1.5B Flow Cell: 1-2
10B Flow Cell: 1-8
25B Flow Cell: 1-8
Index
Yes
The Index 1 (i7) index sequence. Format is a sequence using ACTG. Required if index cycles are specified for the sample in the OverrideCycles.
Must adhere to the following requirements:
Can only contain A, C, G, or T
Length of string must match number of first index cycles in RunInfo.xml or the number specified in OverrideCycles
A value of NA must be used if:
No indexes are specified in OverrideCycles for a sample
Index2
No
See description of Index, applied to Index2.
Sample_Project
No
DRAGEN Germline
DragenGermline_Settings
SoftwareVersion
Yes
The version of the DRAGEN software used to process the DragenGermline pipeline, including conversion to FASTQ. Specified using all three integers included in the DRAGEN version name. For example 4.1.5.
AppVersion
Yes
The version of the workflow-specific application (i.e., DRAGEN Germline), using all three integers included in the version name. For example, 1.0.0.
KeepFastQ
Yes
Select whether FASTQs are saved (true) or discarded (false).
MapAlignOutFormat
Yes
Formatting of the output files. Accepted values are bam, cram, or none. Selecting none produces no map/align output. If MapAlignOutFormat is None, VariantCallingMode cannot be None for any sample
DragenGermline_Data
ReferenceGenomeDir
Yes
Genome name consisting of alphanumeric string with "_" or "-" or ".".
VariantCallingMode
Yes
Variant calling mode for the run.
Accepted values are None, SmallVariantCaller, AllVariantCallers. The option for all variant callers includes Small, Structural, CNV, Repeat Expansions, ROH, CYP2D6, CYP2B6, CYP21A2, SMN, GBA, LPA, RH, and SMN (silent carrier).
QcCoverage1BedFile
No
File name in text (*.txt) or gzip (*.gz) format. Must include the prefix DragenGermline/. Alphanumeric string with underscores (_) or dashes (-) or periods (.) with no spaces allowed. If QcCoverage1BedFile exists in the Data section, but a file is not provided, a value of na must be specified.
QcCoverage2BedFile
No
File name in text (*.txt) or gzip (*.gz) format. Must include the prefix DragenGermline/. Alphanumeric string with underscores (_) or dashes (-) or periods (.) with no spaces allowed. If QcCoverage2BedFile exists in the Data section, but a file is not provided, a value of na must be specified.
QcCoverage3BedFile
No
File name in text (*.txt) or gzip (*.gz) format. Must include the prefix DragenGermline/. Alphanumeric string with underscores (_) or dashes (-) or periods (.) with no spaces allowed. If QcCoverage3BedFile exists in the Data section, but a file is not provided, a value of na must be specified.
QcCrossContaminationVcfFile
No
File name in text (*.txt) or gzip (*.gz) format. Must include the prefix DragenGermline/. Alphanumeric string with underscores (_) or dashes (-) or periods (.) with no spaces allowed. If QcCrossContaminationVcfFile exists in the Data section, but a file is not provided, a value of na must be specified.
Sample_ID
Yes
See description in the BCL Convert section
DRAGEN Enrichment
DragenEnrichment_Settings
SoftwareVersion
Yes
The version of the DRAGEN software used to process the DragenEnrichment pipeline, including conversion to FASTQ. Specified using all three integers included in the DRAGEN version name. For example 4.1.5.
AppVersion
Yes
The version of the workflow-specific application (i.e., DRAGEN Enrichment), using all three integers included in the version name. For example, 1.0.0.
KeepFastQ
Yes
Select whether FASTQs are saved (true) or discarded (false).
MapAlignOutFormat
Yes
Formatting of the output files. Accepted values are bam, cram, or none. Selecting none produces no map/align output. If MapAlignOutFormat is None, VariantCallingMode cannot be None for any sample
DragenEnrichment_Data
ReferenceGenomeDir
Yes
Genome name consisting of alphanumeric string with "_" or "-" or ".".
Bedfile
Conditionally required
BED file to be used for analysis in text (*.txt) or gzip (*.gz) format. Only required if VariantCallingMode is not None. Must include the prefix DragenEnrichment/ before the BED file name. Alphanumeric string with _ or - or . with no spaces allowed. The value must be na if BedFile is placed in the Data section and VariantCallingMode = None.
GermlineOrSomatic
Yes
Accepted values are germline or somatic.
AuxNoiseBaselineFile
No
Alphanumeric string with "_" or "-" or ".". and no spaces in text (*.txt) or gzip (*.gz) format. Applicable only if GermlineOrSomatic is Somatic and VariantCallingMode is not None.
The value must be na if AuxNoiseBaselineFile is placed in the Data section and no AuxNoiseBaselineFile is provided for the sample."
AuxCnvPanelOfNormalsFile
No
Alphanumeric string with "_" or "-" or "." and no spaces in text (*.txt) or gzip (*.gz) format. Optional if VariantCallingMode is AllVariantCallers.
The value must be na if AuxCnvPanelOfNormalsFile is placed in the Data section and no AuxCnvPanelOfNormalsFile is provided for the sample.
VariantCallingMode
Yes
Variant calling mode for the run.
Accepted values are None, SmallVariantCaller, AllVariantCallers. The option for all variant callers includes Small, Structural, and CNV callers (if panel of normals is provided).
AuxCnvPopBAlleleVcfFile
No
Alphanumeric string with _ or - or . with no spaces allowed. Text or gzip format. Optional only if VariantCallingMode = AllVariantCallers and GermlineOrSomatic=somatic. Error otherwise. A note should be added to the UI to indicate that CNV output will only be generated if this file is provided.
AuxGermlineTaggingFile
No
Alphanumeric string with _ or - or . with no spaces allowed. Binary file with .bin extension. Optional when SmallVariantCaller or AllVariantCaller option selected and GermlineOrSomatic=somatic. Error otherwise.
Sample_ID
Yes
See description in the BCL Convert section
DRAGEN RNA
DragenRna_Settings
SoftwareVersion
Yes
The version of the DRAGEN software used to process the DragenRna pipeline, including conversion to FASTQ. Specified using all three integers included in the DRAGEN version name. For example 4.1.5.
AppVersion
Yes
The version of the workflow-specific application (i.e., DRAGEN RNA), using all three integers included in the version name. For example, 1.0.0.
MapAlignOutFormat
Yes
Formatting of the output files. Accepted values are bam, cram, or none. Selecting none produces no map/align output. Selecting none is not allowed if RnaPipelineMode is set to FulPipeline for any sample.
KeepFastQ
Yes
Select whether FASTQs are saved (true) or discarded (false).
DifferentialExpressionEnable
No
Accepted values are true or false. If DifferentialExpressionEnable is true, then RnaPipelineMode must be FullPipeline
DragenRna_Data
ReferenceGenomeDir
Yes
Genome name consisting of alphanumeric string with "_" or "-" or ".". Can be placed in either settings or data section.
RnaGeneAnnotationFile
No
Genotype reference file. Alphanumeric string with "_" or "-" or "." with no spaces allowed. If DifferentialExpressionEnable is True., the GTF file must be provided by the user or included with the reference genome.
RnaPipelineMode
Yes
Accepted values are MapAlign or FullPipeline. The full pipeline option includes quantification and fusion detection.
DownSampleNumReads
No
Specifies the number of fragments to downsample to. For paired-end sequencing, the number of reads at the down-sampling output will be twice the number of fragments specified. Accepted values are integers. If DownSampleNumReads is placed in the Data section, and downsampling is not desired for that sample, a value of na must be used.
Sample_ID
Yes
See description in the BCL Convert section
Comparison1
No
Accepted values are control, comparison, or na. Use only if DifferentialExpressionEnable is true. If RNAPipelineMode is MapAlign, this value must be na. Must have at least 2 control and 2 comparison samples. May have a max of 15 control or comparison samples. All control and comparison samples must have FullPipeline for RnaPipelineMode value and have the same ReferenceGenomeDir and RnaGeneAnnotationFile values.
Comparison2
No
See Comparison1 Description.
Comparison3
No
See Comparison1 Description.
Comparison4
No
See Comparison1 Description.
Comparison5
No
See Comparison1 Description.
DRAGEN Somatic
DragenSomatic_Settings
SoftwareVersion
Required
The version of the DRAGEN software used to process the DragenSomatic pipeline, including conversion to FASTQ. Specified using all three integers included in the DRAGEN version name. For example 4.1.5.
AppVersion
Required
The version of the workflow-specific application (i.e., DRAGEN Somatic), using all three integers included in the version name. For example, 1.0.0.
MapAlignOutFormat
Required
Formatting of the output files. Accepted values are bam, cram, or none. Selecting none produces no map/align output.
KeepFastq
Required
Select whether FASTQs are saved (true) or discarded (false).
DragenSomatic_Data
AuxNoiseBaselineFile
Optional
Alphanumeric string with "_" or "-" or ".". and no spaces in text (*.txt) or gzip (*.gz) format. Applicable only if GermlineOrSomatic is Somatic and VariantCallingMode is not None.
The value must be na if AuxNoiseBaselineFile is placed in the Data section and no AuxNoiseBaselineFile is provided for the sample."
AuxSvNoiseBaselineFile
Optional
Alphanumeric string with "_" or "-" or "." and no spaces in text (*.txt) or gzip (*.gz) format. Optional if VariantCallingMode is AllVariantCallers.
The value must be na if AuxSvNoiseBaselineFile is placed in the Data section and no AuxSvNoiseBaselineFile is provided for the sample.
AuxCnvPopBAlleleVcfFile
Optional
Alphanumeric string with "_" or "-" or "." and no spaces in text (*.txt) or gzip (*.gz) format. Optional if VariantCallingMode is AllVariantCallers.
The value must be na if AuxCnvPopBAlleleVcfFile is placed in the Data section and no AuxCnvPopBAlleleVcfFile is provided for the sample. CNV output will only be generated if this file is provided.
AuxGermlineTaggingFile
Optional
Alphanumeric string with "_" or "-" or "." and no spaces in text (*.txt) or gzip (*.gz) format. Optional if VariantCallingMode is AllVariantCallers.
The value must be na if AuxGermlineTaggingFile is placed in the Data section and no AuxGermlineTaggingFile is provided for the sample. Germline tagging output will only be generated if this file is provided.
VariantCallingMode
Required
Variant calling mode for the run.
Accepted values are None, SmallVariantCaller, AllVariantCallers. The option for all variant callers includes Small, Structural, and CNV callers (if panel of normals is provided).
Sample_ID
Required
See description in the BCL Convert section
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DRAGEN Methylation
DragenMethylation_Settings
SoftwareVersion
Required
The version of the DRAGEN software used to process the DragenMethylation pipeline, including conversion to FASTQ. Specified using all three integers included in the DRAGEN version name. For example 4.1.5.
AppVersion
Required
The version of the workflow-specific application (i.e., DRAGEN Methylation), using all three integers included in the version name. For example, 1.0.0.
MapAlignOutFormat
Required
Formatting of the output files. Accepted values are bam, cram, or none. Selecting none produces no map/align output.
KeepFastq
Required
Select whether FASTQs are saved (true) or discarded (false).
UsesTaps
Required
Select whether the TAPS assay, which directly converts methylated C to T, is used (true) or not used (false)
DragenMethylation_Data
ReferenceGenomeDir
Required
Genome name consisting of alphanumeric string with "_" or "-" or ".". Can be placed in either settings or data section.
MethylationProtocol
Required
Select the library protocol for methylation analysis from the set {'directional’, ‘non-directional’, ‘directional-complement’,‘pbat’}
Sample_ID
Required
See description in the BCL Convert section
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