NovaSeq X Series Settings

For descriptions of each field, including the specific requirements and if the expected value is an integer or string, refer to Parameters.

Standalone Sections

Header Sections

Parameter Required Instrument-Specific Requirements

Parameter	Required	Instrument-Specific Requirements
FileFormatVersion	Yes
RunName	Yes
RunDescription	No
InstrumentType	No
InstrumentPlatform	No	Enter `NovaSeqXSeries` for NovaSeq X Series.
Custom_*	No

FileFormatVersion

Yes

RunName

Yes

RunDescription

InstrumentType

InstrumentPlatform

Enter NovaSeqXSeries for NovaSeq X Series.

Custom_*

Reads Sections

Parameter	Required	Instrument-Specific Requirements
Read1Cycles	Yes
Read2Cycles	No¹
Index1Cycles	No¹	Value must be in the 6–12 range for the following run types: Runs planned in the cloud Runs planned locally using import sample sheet Manual runs with local analysis The total number of read cycles and index cycles cannot exceed the number of cycles specified by the reagent kit. The index cycle limit applies to cycles used as an index, not UMI cycles or trimmed reads.
Index2Cycles	No¹	Value must be in the 6–12 range for the following run types Runs planned in the cloud Runs planned locally using import sample sheet Manual runs with local analysis The total number of read cycles and index cycles cannot exceed the number of cycles specified by the reagent kit. The index cycle limit applies to cycles used as an index, not UMI cycles or trimmed reads.

Parameter

Required

Instrument-Specific Requirements

Read1Cycles

Yes

Read2Cycles

No¹

Index1Cycles

No¹

Value must be in the 6–12 range for the following run types:

Runs planned in the cloud
Runs planned locally using import sample sheet
Manual runs with local analysis

The total number of read cycles and index cycles cannot exceed the number of cycles specified by the reagent kit. The index cycle limit applies to cycles used as an index, not UMI cycles or trimmed reads.

Index2Cycles

No¹

Value must be in the 6–12 range for the following run types

Runs planned in the cloud
Runs planned locally using import sample sheet
Manual runs with local analysis

¹ Depending on the run or library prep kit, this parameter might be required.

Sequencing Sections

Parameter	Required	Instrument-Specific Requirements
CustomRead1Primer	No¹
CustomRead2Primer	No¹
CustomIndex1Primer	No¹
CustomIndex2Primer	No¹

Parameter

Required

Instrument-Specific Requirements

CustomRead1Primer

No¹

CustomRead2Primer

No¹

CustomIndex1Primer

No¹

CustomIndex2Primer

No¹

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

Parameter Required Section Instrument-Specific Requirements

Parameter	Required	Section	Instrument-Specific Requirements
SoftwareVersion	Yes	Settings
FastqCompressionFormat	Yes	Settings	Supported values are `gzip` or `dragen`. To use DRAGEN Illumina Complete Long Reads WGS (ICLR WGS), the recommended value is `gzip`.
AdapterRead1	No	Data	Possible values are a concatenation from the set [A,C,T,G]. NovaSeq X Series also allows `na`. A value of `na` (case insensitive) must be used if both the following statements are accurate: The AdapterRead1 field is placed in the Data section of the Sample Sheet No adapter trimming is desired for the sample.
AdapterRead2	No	Data	Possible values are a concatenation from the set [A,C,T,G]. NovaSeq X Series also allows `na`. A value of na (case insensitive) must be used if both the following statements are accurate: No adapter trimming is desired for the sample. The AdapterRead2 field is placed in the Data section of the Sample Sheet
BarcodeMismatchesIndex1	No	Data	NovaSeq X Series allows the value `na` in addition to the values 0, 1, or 2. Enter a value of na 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	Data	NovaSeq X Series allows the value `na` in addition to the values 0, 1, or 2. Enter a value of na if: BarcodeMismatchesIndex2 exists in the Data section of the Sample Sheet, and OverrideCycles exist in the data section, and Index 2 is masked out for the sample in the OverrideCycles
OverrideCycles	No	Data	OverrideCycles example: `Y151;I8N2;N2I8;Y151` For DRAGEN ICLR WGS, use the following guidelines: When using only DRAGEN BCL Convert and DRAGEN ICLR WGS, enter `Y151;Y151.` When using DRAGEN BCL Convert, DRAGEN ICLR WGS, and additional apps, enter `Y151;NX;NX;Y151,` replacing X with the correct index length.
Sample_ID	Yes	Data
Lane	Yes	Data	For NovaSeq X, 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	Data	For NovaSeq X, a value of na must be used if no indexes are specified in OverrideCycles for a sample. When using DRAGEN ICLR WGS, leave blank.
Index2	No	Data	Always write index sequences in the forward orientation. BCL Convert handles index orientation. When using DRAGEN ICLR WGS, leave blank.

SoftwareVersion

Yes

Settings

FastqCompressionFormat

Yes

Settings

Supported values are gzip or dragen.

To use DRAGEN Illumina Complete Long Reads WGS (ICLR WGS), the recommended value is gzip.

AdapterRead1

Data

Possible values are a concatenation from the set [A,C,T,G]. NovaSeq X Series also allows na.

A value of na (case insensitive) must be used if both the following statements are accurate:

The AdapterRead1 field is placed in the Data section of the Sample Sheet
No adapter trimming is desired for the sample.

AdapterRead2

Data

Possible values are a concatenation from the set [A,C,T,G]. NovaSeq X Series also allows na.

A value of na (case insensitive) must be used if both the following statements are accurate:

No adapter trimming is desired for the sample.
The AdapterRead2 field is placed in the Data section of the Sample Sheet

BarcodeMismatchesIndex1

Data

NovaSeq X Series allows the value na in addition to the values 0, 1, or 2.

Enter a value of na 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

Data

NovaSeq X Series allows the value na in addition to the values 0, 1, or 2.

Enter a value of na if:

BarcodeMismatchesIndex2 exists in the Data section of the Sample Sheet, and
OverrideCycles exist in the data section, and
Index 2 is masked out for the sample in the OverrideCycles

OverrideCycles

Data

OverrideCycles example:

Y151;I8N2;N2I8;Y151

For DRAGEN ICLR WGS, use the following guidelines:

When using only DRAGEN BCL Convert and DRAGEN ICLR WGS, enter Y151;Y151.
When using DRAGEN BCL Convert, DRAGEN ICLR WGS, and additional apps, enter Y151;NX;NX;Y151, replacing X with the correct index length.

Sample_ID

Yes

Data

Lane

Yes

Data

For NovaSeq X, 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

Data

For NovaSeq X, a value of na must be used if no indexes are specified in OverrideCycles for a sample.

When using DRAGEN ICLR WGS, leave blank.

Index2

Data

Always write index sequences in the forward orientation. BCL Convert handles index orientation.

When using DRAGEN ICLR WGS, leave blank.

DRAGEN Enrichment

Parameter Required Instrument-Specific Requirements

Parameter	Required		Instrument-Specific Requirements
SoftwareVersion	Yes	Settings
AppVersion	Yes	Settings
KeepFastQ	Yes	Settings
MapAlignOutFormat	Yes	Settings	If MapAlignOutFormat is set to `none`, VariantCallingMode cannot be `none` for any sample.
ReferenceGenomeDir	Yes	Data
Bedfile	Conditionally required	Data	Only required if `VariantCallingMode` is not `None`. Must include the prefix `DragenEnrichment/` before the BED file name. The value must be `na` if BedFile is placed in the Data section and VariantCallingMode is set to `None`.
GermlineOrSomatic	Yes	Data
AuxNoiseBaselineFile	No	Data	The value must be `na` if AuxNoiseBaselineFile is placed in the Data section and no AuxNoiseBaselineFile is provided for the sample.
AuxCnvPanelOfNormalsFile	Conditionally required	Data	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	Data
Sample_ID	Yes	Data

SoftwareVersion

Yes

Settings

AppVersion

Yes

Settings

KeepFastQ

Yes

Settings

MapAlignOutFormat

Yes

Settings

If MapAlignOutFormat is set to none, VariantCallingMode cannot be none for any sample.

ReferenceGenomeDir

Yes

Data

Bedfile

Conditionally required

Data

Only required if VariantCallingMode is not None.

Must include the prefix DragenEnrichment/ before the BED file name.

The value must be na if BedFile is placed in the Data section and VariantCallingMode is set to None.

GermlineOrSomatic

Yes

Data

AuxNoiseBaselineFile

Data

The value must be na if AuxNoiseBaselineFile is placed in the Data section and no AuxNoiseBaselineFile is provided for the sample.

AuxCnvPanelOfNormalsFile

Conditionally required

Data

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

Data

Sample_ID

Yes

Data

DRAGEN Germline

Parameter Required Instrument-Specific Requirements

Parameter	Required		Instrument-Specific Requirements
SoftwareVersion	Yes	Settings
AppVersion	Yes	Settings
KeepFastQ	Yes	Settings
MapAlignOutFormat	Yes	Settings	If MapAlignOutFormat is set to `none`, VariantCallingMode cannot be `none` for any sample.
ReferenceGenomeDir	Yes	Data
VariantCallingMode	Yes	Data
QcCoverage1BedFile	No	Data	Only supported for v4.1.23 and later.
QcCoverage2BedFile	No	Data	Only supported for v4.1.23 and later.
QcCoverage3BedFile	No	Data	Only supported for v4.1.23 and later.
QcCrossContaminationVcfFile	No	Data	Only supported for v4.1.23 and later.
Sample_ID	Yes	Data

SoftwareVersion

Yes

Settings

AppVersion

Yes

Settings

KeepFastQ

Yes

Settings

MapAlignOutFormat

Yes

Settings

If MapAlignOutFormat is set to none, VariantCallingMode cannot be none for any sample.

ReferenceGenomeDir

Yes

Data

VariantCallingMode

Yes

Data

QcCoverage1BedFile

Data

Only supported for v4.1.23 and later.

QcCoverage2BedFile

Data

Only supported for v4.1.23 and later.

QcCoverage3BedFile

Data

Only supported for v4.1.23 and later.

QcCrossContaminationVcfFile

Data

Only supported for v4.1.23 and later.

Sample_ID

Yes

Data

DRAGEN RNA

Parameter Required Section Instrument-Specific Requirements

Parameter	Required	Section	Instrument-Specific Requirements
SoftwareVersion	Yes	Settings
AppVersion	Yes	Settings
MapAlignOutFormat	Yes	Settings	For the DRAGEN RNA application, selecting none is not allowed if RnaPipelineMode is set to `MapAlign` for any sample.
KeepFastQ	Yes	Settings
DifferentialExpressionEnable	No	Settings
ReferenceGenomeDir	Yes	Data
RnaGeneAnnotationFile	No	Data
RnaPipelineMode	Yes	Data
DownSampleNumReads	No	Data
Sample_ID	Yes	Data
Comparison1	No	Data
Comparison2	No	Data
Comparison3	No	Data
Comparison4	No	Data
Comparison5	No	Data

SoftwareVersion

Yes

Settings

AppVersion

Yes

Settings

MapAlignOutFormat

Yes

Settings

For the DRAGEN RNA application, selecting none is not allowed if RnaPipelineMode is set to MapAlign for any sample.

KeepFastQ

Yes

Settings

DifferentialExpressionEnable

Settings

ReferenceGenomeDir

Yes

Data

RnaGeneAnnotationFile

Data

RnaPipelineMode

Yes

Data

DownSampleNumReads

Data

Sample_ID

Yes

Data

Comparison1

Data

Comparison2

Data

Comparison3

Data

Comparison4

Data

Comparison5

Data

DRAGEN Somatic

Parameter	Required	Section
SoftwareVersion	Yes	Settings
AppVersion	Yes	Settings
KeepFastQ	Yes	Settings
MapAlignOutFormat	Yes	Settings
ReferenceGenomeDir	Yes	Data
AuxNoiseBaselineFile	No	Data
AuxSvNoiseBaselineFile	No	Data
AuxCnvPopBAlleleVcfFile	No	Data
AuxGermlineTaggingFile	No	Data
VariantCallingMode	Yes	Data
Sample_ID	Yes	Data

Parameter

Required

Section

Instrument-Specific Requirements

SoftwareVersion

Yes

Settings

AppVersion

Yes

Settings

KeepFastQ

Yes

Settings

MapAlignOutFormat

Yes

Settings

ReferenceGenomeDir

Yes

Data

AuxNoiseBaselineFile

Data

AuxSvNoiseBaselineFile

Data

AuxCnvPopBAlleleVcfFile

Data

AuxGermlineTaggingFile

Data

VariantCallingMode

Yes

Data

Sample_ID

Yes

Data

DRAGEN Methylation

Parameter	Required	Section
SoftwareVersion	Yes	Settings
AppVersion	Yes	Settings
KeepFastQ	Yes	Settings
MapAlignOutFormat	Yes	Settings
UsesTaps	Yes	Settings
ReferenceGenomeDir	Yes	Data
MethylationProtocol	No	Data
Sample_ID	No	Data

Parameter

Required

Section

Instrument-Specific Requirements

SoftwareVersion

Yes

Settings

AppVersion

Yes

Settings

KeepFastQ

Yes

Settings

MapAlignOutFormat

Yes

Settings

UsesTaps

Yes

Settings

ReferenceGenomeDir

Yes

Data

MethylationProtocol

Data

Sample_ID

Data

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