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How to set up analysis for AmpliSeq for Illumina libraries with off instrument Local Run Manager 2

With the launch of AmpliSeq for Illumina products, DNA Amplicon and RNA Amplicon analysis modules for Local Run Manager 2 have been released.* Local Run Manager 2 is available for off-instrument analysis of data from iSeq, MiniSeq, MiSeq, and NextSeq 500/550 systems, and onboard the iSeq and MiSeqs running MiSeq Control Software version 3.

For instruments without on-instrument Local Run Manager 2, analysis must be performed using an off-instrument installation.
For MiniSeq users, refer to the How to set up an AmpliSeq for Illumina run using Local Run Manager for MiniSeq bulletin.

After sequencing has finished, transfer the complete run folder to a computer where off-instrument Local Run Manager 2 with AmpliSeq modules is installed. Note: Run data must be copied to the local computer. Local Run Manager may fail to import runs from locations in user-specific folders, such as Desktop and Documents. Analysis of run data from a network location is not recommended. Users performing DNA Amplicon analysis must also download and install the version of the hg19 human genome available on the Local Run Manager support site.

To analyze data with Local Run Manager 2 off-instrument, first create the run either manually or by importing a sample sheet:

Option 1, manual run setup:

Select Create Run from the Local Run Manager dashboard and select either DNA Amplicon or RNA Amplicon.
Enter Run name and select AmpliSeq Library PLUS for Illumina (96) from the Library Kit dropdown menu.
Choose the appropriate Module-Specific Settings required for analysis. For more information, refer to the DNA Amplicon and RNA Amplicon analysis module guides.

When performing DNA Amplicon analysis, AmpliSeq for Illumina libraries are aligned using the BWA Whole-Genome aligner. The TruSeq Amplicon aligner is for TruSeq Amplicon libraries only.

Indel realignment is an optional setting. Using this option may improve detection of medium size indels, but changes to overall accuracy may depend on specific panels. Total analysis time will increase when indel realignment option is selected. If you would like to turn on this option: 1. Expand Show Advanced Module Settings

2. Select + Add custom setting    
  ![](./000001319_files/image2.jpg)    

3. Enter **VariantCallerRealignIndels** in the field “Type custom setting here”  
4. enter **1** for true in the field “Type setting value here” to enable the Indel realignment option.    
  ![](./000001319_files/image3.jpg)

Enter Sample IDs in the sample table and choose the index plate well from the dropdown menu. The contents of each well can be found in the Index Adapters Pooling Guide. Index sequences will populate automatically.
Import or select the manifest from the Manifest dropdown menu and select the reference genome from Genome dropdown menu.
Select Save Run to return to the Local Run Manager dashboard or Export Sample Sheet to save a copy of the run configuration for future use.

Option 2, set up with sample sheet:

Sample sheets can be created using Illumina Experiment Manager 1.15 or later or using Local Run Manager. When using Illumina Experiment Manager, select MiSeq as the instrument and FASTQ Only as the application.

Note: Local Run Manager will automatically reverse complement the second index when performing analysis of MiniSeq and NextSeq 500/550 data. The forward (MiSeq) orientation of indexes should always be used.

Select Create Run from the Local Run Manager dashboard.
From the dropdown menu Select DNA Amplicon or RNA Amplicon from the Local Run Manager dashboard.
Enter a Run name if the Experiment Name was not specified in the sample sheet.
Follow steps 3-5 of option 1 to choose module-specific settings, the manifest, and the reference genome.
Select Save Run to return to the Local Run Manager dashboard or Export Sample Sheet to save a copy of the run configuration for future use.

The run will now appear as Ready for Sequencing. To begin the analysis:

Select More Options (the three grey dots on the right of the run name) and select Import.
In Import Run window, enter the path to the location of the run folder.
Enter the location of the output folder where the analysis results will be saved. The output can be a local drive or a network location using the full UNC path. Mapped letter drives are not supported as network output locations.
Select Import Run. The analysis will start automatically.

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For instruments without on-instrument Local Run Manager 2, analysis must be performed using an off-instrument installation.
For MiniSeq users, refer to the How to set up an AmpliSeq for Illumina run using Local Run Manager for MiniSeq bulletin.

To analyze data with Local Run Manager 2 off-instrument, first create the run either manually or by importing a sample sheet:

Option 1, manual run setup:

Select Create Run from the Local Run Manager dashboard and select either DNA Amplicon or RNA Amplicon.
Enter Run name and select AmpliSeq Library PLUS for Illumina (96) from the Library Kit dropdown menu.
Choose the appropriate Module-Specific Settings required for analysis. For more information, refer to the DNA Amplicon and RNA Amplicon analysis module guides.

When performing DNA Amplicon analysis, AmpliSeq for Illumina libraries are aligned using the BWA Whole-Genome aligner. The TruSeq Amplicon aligner is for TruSeq Amplicon libraries only.

2. Select + Add custom setting    
  ![](./000001319_files/image2.jpg)    

3. Enter **VariantCallerRealignIndels** in the field “Type custom setting here”  
4. enter **1** for true in the field “Type setting value here” to enable the Indel realignment option.    
  ![](./000001319_files/image3.jpg)

Enter Sample IDs in the sample table and choose the index plate well from the dropdown menu. The contents of each well can be found in the Index Adapters Pooling Guide. Index sequences will populate automatically.
Import or select the manifest from the Manifest dropdown menu and select the reference genome from Genome dropdown menu.
Select Save Run to return to the Local Run Manager dashboard or Export Sample Sheet to save a copy of the run configuration for future use.

Option 2, set up with sample sheet:

Sample sheets can be created using Illumina Experiment Manager 1.15 or later or using Local Run Manager. When using Illumina Experiment Manager, select MiSeq as the instrument and FASTQ Only as the application.

Note: Local Run Manager will automatically reverse complement the second index when performing analysis of MiniSeq and NextSeq 500/550 data. The forward (MiSeq) orientation of indexes should always be used.

Select Create Run from the Local Run Manager dashboard.
From the dropdown menu Select DNA Amplicon or RNA Amplicon from the Local Run Manager dashboard.
Select Import Sample Sheet.
Enter a Run name if the Experiment Name was not specified in the sample sheet.
Follow steps 3-5 of option 1 to choose module-specific settings, the manifest, and the reference genome.
Select Save Run to return to the Local Run Manager dashboard or Export Sample Sheet to save a copy of the run configuration for future use.

The run will now appear as Ready for Sequencing. To begin the analysis:

Select More Options (the three grey dots on the right of the run name) and select Import.
In Import Run window, enter the path to the location of the run folder.
Enter the location of the output folder where the analysis results will be saved. The output can be a local drive or a network location using the full UNC path. Mapped letter drives are not supported as network output locations.
Select Import Run. The analysis will start automatically.

For any feedback or questions regarding this article (Illumina Knowledge Article #1319), contact Illumina Technical Support .