NextSeq 1000/2000 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 NextSeq1k2k for NextSeq 1000/2000 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 NextSeq 2000, populate the field with NextSeq2000.
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. Enter NextSeq 1000 or NextSeq 2000.
RunName
No
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. Enter NextSeq 1000 or NextSeq 2000.
Reads Sections
Index1Cycles
No
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
No
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.
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
No
Identifies the library prep kit used for the run.
String with ASCII characters except for * and the control characters
CRandLF.Only one library prep kit is allowed.
In the NextSeq 1000/2000 Control Software v1.3 or later, the required custom recipe is automatically selected if one of the following kits is specified as the library prep kit.
Illumina Stranded Total RNA Prep with Ribo-Zero Plus kit
Illumina Stranded mRNA Prep kit
Enter one of the following values.
For Illumina Stranded Total RNA Prep with Ribo-Zero Plus kit, enter ILMNStrandedTotalRNA.
For Illumina Stranded mRNA Prep kit, enter ILMNStrandedmRNA.
¹ Depending on the run or library prep kit, this parameter might be required.
Application Sections
BCL Convert
BCLConvert_Settings
SoftwareVersion
Required
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.
AdapterRead1
Optional
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
Optional
See description of AdapterRead1, applied to AdapterRead2.
BarcodeMismatchesIndex1
Optional
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
Optional
See description of BarcodeMismatchesIndex1, applied to BarcodeMismatchesIndex2.
OverrideCycles
Optional
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 by both indexes together 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 10 bases toward the limit of 27
"I4N1I3;I5N3": counts as (8+5) 13 bases toward the limit of 27
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;Y151Y151;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.
FastqCompressionFormat
Optional
The compression format for the FASTQ output files. Allowed values are gzip or dragen.
NoLaneSplitting
Optional
If set to true, output all lanes of a flow cell to the same FASTQ files consecutively.
BCLConvert_Data
Sample_ID
Required
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
Index
Optional
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
Optional
See description of Index, applied to Index2.
DRAGEN Germline
DragenGermline_Settings
SoftwareVersion
Required
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.
ReferenceGenomeDir
Required
Genome name consisting of alphanumeric string with "_" or "-" or ".".
MapAlignOutFormat
Optional
Formatting of the output files. Accepted values are bam, cram, or none. Default is bam.
KeepFastQ
Optional
Select whether FASTQs are saved (true) or discarded (false).
DragenGermline_Data
Sample_ID
Required
See description in the BCL Convert section
DRAGEN Enrichment
DragenEnrichment_Settings
SoftwareVersion
Required
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.
MapAlignOutFormat
Optional
Formatting of the output files. Accepted values are bam, cram, or none. Default is bam.
ReferenceGenomeDir
Required
Genome name consisting of alphanumeric string with "_" or "-" or ".".
Bedfile
Required
BED file to be used for analysis in text (*.txt) or gzip (*.gz) format. Alphanumeric string with _ or - or . with no spaces allowed.
KeepFastQ
Optional
Select whether FASTQs are saved (true) or discarded (false).
GermlineOrSomatic
Required
Indicator of pipeline type. Accepted values are germline or somatic.
AuxNoiseBaselineFile
Optional
Alphanumeric string with "_" or "-" or ".". and no spaces in text (*.txt) or gzip (*.gz) format. Applicable only if GermlineOrSomatic is Somatic.
AuxCnvPanelOfNormalsFile
Optional
Alphanumeric string with "_" or "-" or "." and no spaces in text (*.txt) or gzip (*.gz) format.
DragenEnrichment_Data
Sample_ID
Required
See description in the BCL Convert section
DRAGEN RNA
DragenRna_Settings
SoftwareVersion
Required
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.
MapAlignOutFormat
Optional
Formatting of the output files. Accepted values are bam, cram, or none. Default is bam.
ReferenceGenomeDir
Required
Genome name consisting of alphanumeric string with "_" or "-" or ".".
RnaGeneAnnotationFile
Optional
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.
KeepFastQ
Optional
Select whether FASTQs are saved (true) or discarded (false).
DifferentialExpressionEnable
Optional
Accepted values are true or false.
DragenRna_Data
Sample_ID
Required
See description in the BCL Convert section
Comparison1
Optional
Accepted values are control, comparison, or na. Use only if DifferentialExpressionEnable is true. Must have at least 2 control and 2 comparison samples. May have a max of 15 control or comparison samples.
Comparison2
Optional
See Comparison1 description.
Comparison3
Optional
See Comparison1 description.
Comparison4
Optional
See Comparison1 description.
Comparison5
Optional
See Comparison1 description.
DRAGEN Single Cell RNA
DragenSingleCellRna_Settings
SoftwareVersion
Required
The version of the DRAGEN software used to process the DragenSingleCellRNA pipeline, including conversion to FASTQ. Specified using all three integers included in the DRAGEN version name. For example 4.1.5.
MapAlignOutFormat
Optional
Formatting of the output files. Accepted values are bam, cram, or none. Default is bam.
ReferenceGenomeDir
Required
Genome name consisting of alphanumeric string with "_" or "-" or ".".
RnaGeneAnnotationFile
Optional
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.
KeepFastQ
Optional
Select whether FASTQs are saved (true) or discarded (false).
BarcodePosition
Required
Text string indicating the location of the bases corresponding to the Barcode within the specified BarcodeRead. Base positions are indexed starting at position zero in the Read. Examples: 0_15, or 0_8+21_29+43_51 if the barcodes are not contiguous.
UmiPosition
Required
Text string indicating the location of the bases corresponding to the UMI within the specified BarcodeRead. Base positions are indexed starting at position zero in the Read. Example: 16_27
BarcodeRead
Required
Text string indicating the location within the sequencing run of the Barcode Read, which contains both the Barcode and the Umi. Readn, where n is from the set { 1, 2 }
BarcodeSequenceWhiteList
Required
List of expected barcode sequences
BarcodeSequenceList
Required
List of expected barcode sequences
RnaLibraryType
Required
Valid values are StrandedForward, StrandedReverse, Unstranded
DragenSingleCellRna_Data
Sample_ID
Required
See description in the BCL Convert section
DRAGEN Amplicon
DragenAmplicon_Settings
SoftwareVersion
Required
The version of the DRAGEN software used to process the DragenAmplicon pipeline, including conversion to FASTQ. Specified using all three integers included in the DRAGEN version name. For example 4.1.5.
MapAlignOutFormat
Optional
Formatting of the output files. Accepted values are bam, cram, or none. Default is bam.
ReferenceGenomeDir
Required
Genome name consisting of alphanumeric string with "_" or "-" or ".".
KeepFastQ
Optional
Select whether FASTQs are saved (true) or discarded (false).
DnaBedFile
Required
BED file to be used for analysis in text (*.txt) or gzip (*.gz) format. Alphanumeric string with _ or - or . with no spaces allowed.
DnaGermlineOrSomatic
Required
Indicator of "germline" or "somatic" type for DNA samples
DragenSingleCellRna_Data
Sample_ID
Required
See description in the BCL Convert section
DnaOrRna
Required
Indicator of a DNA or RNA sample. Valid values are dna and rna.
DRAGEN GeoMxNGS
DragenGeoMxNGS_Settings
SoftwareVersion
Required
The version of the DRAGEN software~~,~~ used to process the DragenGeoMxNGS pipeline, including conversion to FASTQ. Specified using all three integers included in the DRAGEN version name. For example 4.1.5.
GeoMxConfigFile
Required
GeoMx configuration file
BarcodeSequenceList
Required
List of expected barcode sequences
KeepFastq
Optional
Select whether FASTQs are saved (true) or discarded (false).
DragenGeoMxNGS_Data
Sample_ID
Required
See description in the BCL Convert section
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