Planning a Manual Mode Run on MiSeq i100 Series

Background

There are two options to set up a run in Manual Mode on the MiSeq i100 Series.

1. Manual Run (Generate BCLs Only) - Select this option if not performing secondary analysis (FASTQ Generation). Sample sheet is optional, though will not trigger Secondary Analysis if attached.

2. Manual Run (Import Sample Sheet) - Select this option to import a sample sheet and perform Secondary Analysis on-instrument.

Start a Manual Run (Generate BCLs Only)

1. Sign in to the Control Software.

2. Select Start.

3. Toggle the slider bar to select Manual.

4. Select the Generate BCL files radio button.

5. Enter a run name. The run name can only contain alphanumeric characters, spaces, dashes, and underscores.

6. Select single- or paired-end for the read type.

7. Enter the number of cycles performed in each read. The total number of read cycles and index cycles cannot exceed the number of cycles specified by the reagent kit. All fields must be filled in, input 0 to skip a specific Read.

   1. Read 1—Enter the number of cycles for Read 1.

   2. Index 1—Enter the length of the index read for Index 1.

   3. Index 2—Enter the length of the index read for Index 2.

   4. Read 2—Enter the number of cycles for Read 2. This value is typically the same as the Read 1 value.

8. [Optional] Select sample sheet.

9. Select Review, and then review the run. Configure the following optional run settings as needed:

   1. If Read First sequencing is required, de-select the Sequence Indexes First checkbox.

   2. If using custom primers, select the appropriate custom primers checkboxes. Custom primers are only available for Read First sequencing.

   3. If the instrument is connected to the cloud and logged in with BaseSpace Sequence Hub account, select a cloud run setting.

   4. To use a different output folder from the default, modify the output folder. Change the default output folder in system settings.

   5. Select a custom recipe file.

10. When finished, refer to Prepare Dry Cartridge.

Start a Manual Run (Import Sample Sheet)

Format Sample Sheet

Before importing sample sheet, the sample sheet must be formatted properly. Create the sample sheet template using the local Run Planning application on the instrument, or in the cloud using BaseSpace Sequence Hub.

• To create a sample sheet template using one of the local DRAGEN applications on the instrument, refer to the steps in the Set Up DRAGEN Secondary Analysis section and select Export sample sheet in the final step.
• To export a sample sheet for a planned run from BaseSpace Sequence Hub, select Export.

Import Sample Sheet

1. Select Start.

2. Sign in to the control software.

3. Using the slider bar, select Manual.

4. Select Import sample sheet for local analysis.

5. Select Select file and open the sample sheet v2 file. Refer to Format Sample Sheet for information on sample sheet formatting and requirements.

   1. If importing a Sample Sheet from a USB storage device, ensure the USB device is plugged into the USB 3.0 port and has been added to External Storage.

   2. See Add a USB Drive for more information.
6. Select Review, and then review the run. Configure the following optional run settings as needed:

   1. If using custom primers, select the appropriate custom primers checkboxes. Refer to Custom Primers for more information.
   2. If Read First sequencing is required, deselect the Sequence Indexes First checkbox.

   3. If the instrument is connected to the cloud, and logged in with BaseSpace Sequence Hub account, select a cloud run setting.

   4. To use a different output folder from the default, modify the output folder. The default output folder is configured in system settings.

   5. Modify the Transfer BCL data folder to the external storage and/or cloud checkbox. The default is to transfer files, unless configured differently in system settings.

   6. Select a custom recipe file.

7. When finished, refer to Prepare Dry Cartridge.

For any feedback or questions regarding this article (Illumina Knowledge Article #9499), contact Illumina Technical Support [e-mail].