Set Up a Sequencing Run (Manual Mode)

Setting up a run in Manual mode means specifying run parameters in the control software and performing analysis off-instrument using an application of your choice. The software saves data to the output folder for the analysis. Generating FASTQ files requires an additional step. If using custom primers, refer to Custom Primers for run setup instructions.

1. If the system is configured for run analysis, collaboration, and storage using BaseSpace Sequence Hub, create a sample sheet for the run:
a. Download the iSeq 100 System Sample Sheet Template for Manual Mode. Refer to the iSeq 100 Sequencing System Software Downloads page on the Illumina support center for more information.
b. Modify the template as needed. Make sure that:
• Index 2 (i5) adapter sequences are in the correct orientation. For orientations, refer to Illumina Adapter Sequences (1000000002694).
• Sample sheet values must match control software values. For example, enter 151 in the Read 1 field of both the sample sheet and the Run Setup screen.
c. Save the template in CSV file format.
2. In the control software, select Sequence.

The software opens the door at an angle, ejects the tray, and initiates the series of run setup screens.

3. [Optional] Select Help to view an onscreen prompt.

Help prompts appear on each screen to provide supplementary guidance.

1. Make sure that the cartridge is thawed and contains the flow cell and diluted library.
2. Place the cartridge onto the tray so that the access window faces up and the flow cell is inside the instrument. Do not push the cartridge or tray into the instrument.

3. Select Close Door to retract the cartridge and close the door.

A panel appears on the left side of the screen to show information from the scanned consumables.

The BaseSpace Sequence Hub screen appears when the system is configured for Run Monitoring or Run Monitoring and Storage.

1. To disconnect the current run from BaseSpace Sequence Hub, select Skip BaseSpace Sequence Hub Sign In.

Instrument performance data are still sent to Illumina.

2. To change connectivity for the current run, select a Configuration option:
• Run Monitoring Only—Send only InterOp files to BaseSpace Sequence Hub to allow remote monitoring.
• Run Monitoring and Storage—Send run data to BaseSpace Sequence Hub to allow remote monitoring and analysis.
3. Enter your BaseSpace Sequence Hub credentials, and then select Sign In.
4. If the Available Workgroups list appears, select a workgroup to upload run data to.

The list appears when you belong to multiple workgroups.

5. Select Run Setup.
1. In the Run Name field, enter a unique name of your preference to identify the current run.

The run name can contain alphanumeric characters, hyphens, and underscores.

2. For Read Type, select one of the following options:
• Single Read—Perform one sequencing read, which is the faster, simpler option.
• Paired End—Perform two sequencing reads, which generates higher-quality data and provides more accurate alignment.
3. For Read Cycle, enter the number of cycles to perform in each read.
• For Read 1 and Read 2, add one cycle to the desired number of cycles.
• For a PhiX-only run, enter 0 in both index fields.

Read

Number of Cycles

Read 1

26–151

Index 1

Up to 10

Index 2

Up to 10

Read 2

26–151

Read 2 is typically the same value as Read 1, including the extra cycle. Index 1 sequences the i7 index adapter, and Index 2 sequences the i5 index adapter.

4. To specify an output folder for the current run or upload a sample sheet, select Advanced:
• In the Output Folder field, enter the path to the output folder location or select Browse and navigate.
• In the Sample Sheet field, enter the path to the sample sheet location or select Browse and navigate.
5. Select Start Run to initiate the pre-run check.

Pre-run checks include an instrument check and a flow check. The flow check pierces the cartridge seals and passes reagent through the flow cell, so consumables cannot be reused after it starts.

1. Wait about 15 minutes for pre-run checks to complete.

The run starts automatically after successful completion. Unless the system is muted, a chime sound indicates that the run has started.

Opening the door during the pre-run check or the run can cause run failure.

2. If an error occurs during the instrument check, select Retry to redo the check.

The instrument check precedes the flow check. When a check is in progress, the bar is animated.

3. If an error recurs, refer to Error Message Resolution to troubleshoot.
1. Monitor run progress and metrics as they appear on the Sequencing screen after cycle 26.

Metric

Description

%Q30 Read 1

The percentage of Read 1 base calls with a Q-score ≥ 30.

%Clusters PF

The percentage of clusters that passed quality filters.

%Occupancy

The percentage of flow cell wells containing clusters.

Projected Total Yield

The expected number of bases called for the run.
2. To monitor file copying and other run processes, select the control software menu, and then select Process Management.
1. When sequencing is complete, select Eject Cartridge.

The software ejects the used cartridge from the instrument.

2. Remove the cartridge from the tray.
3. Remove the flow cell from the cartridge.
4. Dispose of the flow cell, which has electronic components, in accordance with applicable standards for your region.
5. Dispose of the cartridge, which contains used reagents, in accordance with applicable standards for your region.

A post-run wash is not necessary because fluidics are discarded with the cartridge.

This set of reagents contains potentially hazardous chemicals. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Ventilation should be appropriate for handling of hazardous materials in reagents. Wear protective equipment, including eye protection, gloves, and laboratory coat appropriate for risk of exposure. Handle used reagents as chemical waste and discard in accordance with applicable regional, national, and local laws and regulations. For additional environmental, health, and safety information, refer to the SDS at support.illumina.com/sds.html.

6. Select Close Door to reload the tray and return to the Home screen.

The software automatically reloads the tray and sensors confirm cartridge removal.