Set up NextSeq 1000/2000 Secondary Analysis
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Configure the settings for the analysis type selected for your run.
Use the following steps to configure Illumina DRAGEN BCL Convert analysis.
1- [Optional] Enter the following optional settings:
AdapterRead1: Adapter sequence for read 1. If using an Illumina library prep kit, leave the AdapterRead1 field empty.
AdapterRead2: Adapter sequence for read 2. If using an Illumina library prep kit, leave the AdapterRead2 field empty.
BarcodeMismatchesIndex1: The number of allowed mismatches between the first index read and index sequence. Allowed values are 0,1, or 2. The default value is 1. If a barcode is 6 bp, the recommended value is 0.
BarcodeMismatchIndex2: The number of allowed mismatches between the second index read and index sequence. Allowed values are 0,1, or 2. The default value is 1. If a barcode is 6 bp, the recommended value is 0.
OverrideCycles: String used to specify UMI cycles and mask out cycles of a read. The following values are allowed:
Y—Specifies a sequencing read.
I—Specifies an indexing read.
U—Specifies a UMI length to be trimmed from the read.
Each element is separated by semicolons. The following is an example of OverrideCycles input: N1Y150;I8;I7N1;Y141U10
2- For Local mode, select whether to save a copy of your FASTQ files. FASTQ files are only generated if you select to keep FASTQ files.
3- For Local mode, select one of the following FASTQ output format options:
gzip - Save the FASTQ files in gzip format
DRAGEN - Save FASTQ files in ora format
4- Review and Complete the run configuration.
To save the run configuration for further editing, select Save as Draft. Draft runs appear in BaseSpace Sequence Hub planned runs list, but will not be available on the instrument until the draft runs are saved as planned.
To send the run configuration to your BaseSpace Sequence Hub account, select Save as Planned. Runs submitted to BaseSpace Sequence Hub appear in the planned runs list and are available for systems using Cloud mode or Hybrid mode.
To save the run configuration as a sample sheet in v2 file format, select Export. The sample sheet is required to initiate runs on systems using Local mode. This option is only available if Local was selected for analysis location.
Use the following steps to configure Illumina DRAGEN Enrichment analysis:
1- Select a reference genome. If using Nextera Flex for Enrichment with the Illumina Exome Panel, only hg19 and hs37d5 reference genomes are compatible with the DRAGEN Enrichment pipeline.
2- Select a *.bed file containing the regions you would like to target or upload a new custom file. Make sure the BED file's reference genome matches the reference genome selected in step 1. For a new custom BED file, use the following naming format: name_of_panel_versionNumber.referencegenome.bed
Local mode — Select Upload Custom File (Local) to upload for a single run or Upload Custom File (BaseSpace) for repeated use.
Cloud or Hybrid mode — Select Upload Custom File (BaseSpace). The custom BED file is only available in the Workgroup it was uploaded to.
3- Select either the germline or somatic variant caller
4- [Optional] If using the somatic variant caller, select a noise baseline file.
If using the DRAGEN Enrichment workflow in somatic mode, you can use a noise baseline file to filter out sequencing or systematic noise. You can download standard custom noise files from the Illumina Support Site or create a custom noise baseline file. Refer to Custom Noise Baseline File for more information.
5- Select a map/align output format.
6- For Local mode, select whether to save a copy of your FASTQ files. FASTQ files are only generated if you select to keep FASTQ files.
7- For Local mode, select one of the following FASTQ output format options:
gzip - Save the FASTQ files in gzip format
DRAGEN - Save FASTQ files in ora format
8- Review and Complete the run configuration.
To save the run configuration for further editing, select Save as Draft. Draft runs appear in BaseSpace Sequence Hub planned runs list, but will not be available on the instrument until the draft runs are saved as planned.
To send the run configuration to your BaseSpace Sequence Hub account, select Save as Planned. Runs submitted to BaseSpace Sequence Hub appear in the planned runs list and are available for systems using Cloud mode or Hybrid mode.
To save the run configuration as a sample sheet in v2 file format, select Export. The sample sheet and secondary analysis supporting files are downloaded in a *.zip folder and are required to initiate runs on systems using Local mode. This option is only available if Local was selected for analysis location.
Use the following steps to configure Illumina DRAGEN Germline analysis:
1- Select a reference genome.
2- Select a map/align output format.
3- For Local mode, select whether to save a copy of your FASTQ files. FASTQ files are only generated if you select to keep FASTQ files.
4- For Local mode, select one of the following FASTQ output format options:
gzip - Save the FASTQ files in gzip format
DRAGEN - Save FASTQ files in ora format
5- Review and Complete the run configuration.
To save the run configuration for further editing, select Save as Draft. Draft runs appear in BaseSpace Sequence Hub planned runs list, but will not be available on the instrument until the draft runs are saved as planned.
To send the run configuration to your BaseSpace Sequence Hub account, select Save as Planned. Runs submitted to BaseSpace Sequence Hub appear in the planned runs list and are available for systems using Cloud mode or Hybrid mode.
To save the run configuration as a sample sheet in v2 file format, select Export. The sample sheet is required to initiate runs on systems using Local mode. This option is only available if Local was selected for analysis location.
Use the following steps to configure Illumina DRAGEN RNA analysis:
1- Select a reference genome.
2- Select your map/align output format.
3- For Local mode, select whether to save a copy of your FASTQ files. FASTQ files are only generated if you select to keep FASTQ files.
4- For Local mode, select one of the following FASTQ output format options:
gzip - Save the FASTQ files in gzip format
DRAGEN - Save FASTQ files in ora format
5- [Optional] Upload a Gene Transfer Format (GTF) file for RNA Annotation file
Local mode — Select Upload Custom File (Local) to upload for a single run or Upload Custom File (BaseSpace) for repeated use.
Cloud or Hybrid mode — Select Upload Custom File (BaseSpace). The GTF file is only available in the Workgroup it was uploaded to.
6- Select whether to enable differential expression.
7- If you enabled differential expression, select a control or comparison value for each sample. In each comparison group, any sample marked as control is compared with all samples marked as comparison. If the sample does not contain a control or comparison value, select na as the value.
8- Review and Complete the run configuration.
To save the run configuration for further editing, select Save as Draft. Draft runs appear in BaseSpace Sequence Hub planned runs list, but will not be available on the instrument until the draft runs are saved as planned.
To send the run configuration to your BaseSpace Sequence Hub account, select Save as Planned. Runs submitted to BaseSpace Sequence Hub appear in the planned runs list and are available for systems using Cloud mode or Hybrid mode.
To save the run configuration as a sample sheet in v2 file format, select Export. The sample sheet and secondary analysis supporting files are downloaded in a *.zip folder, and are required to initiate runs on systems using Local mode. This option is only available if Local was selected for analysis location.
Use the following steps to configure Illumina DRAGEN Single Cell RNA analysis:
1- Select a reference genome.
2- [Optional] Upload a Gene Transfer Format (GTF) file for RNA Annotation file
Local mode — Select Upload Custom File (Local) to upload for a single run or Upload Custom File (BaseSpace) for repeated use.
Cloud or Hybrid mode — Select Upload Custom File (BaseSpace). The GTF file is only available in the Workgroup it was uploaded to.
3- Select your map/align output format.
4- For Local mode, select whether to save a copy of your FASTQ files. FASTQ files are only generated if you select to keep FASTQ files.
5- For Local mode, select one of the following FASTQ output format options:
gzip - Save the FASTQ files in gzip format
DRAGEN - Save FASTQ files in ora format
6- Select the configuration identical to your library prep kit type. For example, if you selected Single Cell RNA Library Kit 1 as your library prep kit, select Type 1 for the Configuration Type.
7- Select the barcode read.
8- [Optional] Edit the number of bases in the barcodes and the UMI. The values are automatically filled based on the library prep kit and configuration type you selected.
9- Select the strand orientation.
10- [Optional] Select a file containing your barcode sequences or upload a new custom file.
11- If using an Advanced/Custom configuration type, enter values for the number of override cycles, the barcode position, and the UMI position.
12- Review and Complete the run configuration.
To save the run configuration for further editing, select Save as Draft. Draft runs appear in BaseSpace Sequence Hub planned runs list, but will not be available on the instrument until the draft runs are saved as planned.
To send the run configuration to your BaseSpace Sequence Hub account, select Save as Planned. Runs submitted to BaseSpace Sequence Hub appear in the planned runs list and are available for systems using Cloud mode or Hybrid mode.
To save the run configuration as a sample sheet in v2 file format, select Export. The sample sheet and secondary analysis supporting files are downloaded in a *.zip folder, and are required to initiate runs on systems using Local mode. This option is only available if Local was selected for analysis location.
Use the following steps to configure Illumina DRAGEN Amplicon analysis:
1- Select a reference genome.
2- Select a *.bed file containing the regions you would like to target or upload a new custom file. Make sure the BED file's reference genome matches the reference genome selected in step 1. For a new custom BED file, use the following naming format: name_of_panel_versionNumber.referencegenome.bed
Local mode — Select Upload Custom File (Local) to upload for a single run or Upload Custom File (BaseSpace) for repeated use.
Cloud or Hybrid mode — Select Upload Custom File (BaseSpace). The custom BED file is only available in the Workgroup it was uploaded to.
3- Select either the germline or somatic variant caller
4- Select a map/align output format.
5- For Local mode, select whether to save a copy of your FASTQ files. FASTQ files are only generated if you select to keep FASTQ files.
6- For Local mode, select one of the following FASTQ output format options:
gzip - Save the FASTQ files in gzip format
DRAGEN - Save FASTQ files in ora format
8- Review and Complete the run configuration.
To save the run configuration for further editing, select Save as Draft. Draft runs appear in BaseSpace Sequence Hub planned runs list, but will not be available on the instrument until the draft runs are saved as planned.
To send the run configuration to your BaseSpace Sequence Hub account, select Save as Planned. Runs submitted to BaseSpace Sequence Hub appear in the planned runs list and are available for systems using Cloud mode or Hybrid mode.
To save the run configuration as a sample sheet in v2 file format, select Export. The sample sheet and secondary analysis supporting files are downloaded in a *.zip folder and are required to initiate runs on systems using Local mode. This option is only available if Local was selected for analysis location.