Generate FASTQ from an incomplete run in Local Run Manager

Local Run Manager is the integrated run-planning and analysis software for the Illumina benchtop sequencers iSeq 100, MiniSeq, MiSeq, and NextSeq. Local Run Manager is also available for off-instrument installation on a separate computer. You can generate FASTQ files from sequencing data from benchtop instruments even if the run does not complete. To generate FASTQs from an incomplete run in Local Run Manager, follow the steps below.

Before you start:* Determine the last completed cycle with a complete set of BCL files in the BaseCalls directory of your run folder. The number of BCL files per cycle will vary by instrument. If the run terminated early because of a run performance issue, review the run in Sequencing Analysis Viewer first to determine the last cycle with good quality base calls.

• If using the onboard Local Run Manager on an instrument, make sure that a sequencing run is not in progress.

• If you are using BaseSpace Sequence Hub for analysis and FASTQ generation instead of Local Run Manager, email [e-mail] Support for assistance.

• If the run stopped before completing the index reads, it is not possible to demultiplex and identify samples and data cannot be obtained.

• Make sure that an RTAcomplete.txt file is not currently present in the run folder.

Steps to take:

1. Determine the last completed and usable cycle. Calculate how many cycles total have been successfully completed and extracted and what the breakdown of cycles is. For example, the original run setup was paired-end 150 cycles with 8 cycle dual index reads, and the last usable cycle is cycle 216. The new run breakdown would be 150 cycles for Read 1, 8 cycles for Index 1, 8 cycles for Index 2, and 50 cycles for Read 2.

2. Back up the original RunInfo.xml file in the run folder, and if present, the original SampleSheet.csv file by copying them and renaming them RunInfo.xml.BAK and SampleSheet.csv.BAK. Using a plain text editor such as Notepad, edit the Reads section of the RunInfo.xml file in the run folder to use the data only up through the last usable cycle. For example:

Original RunInfo.xml:

Edited RunInfo.xml:

Note: If the run did not complete Read 1, sample data cannot be demultiplexed, but can still generate a non-demultiplexed FASTQ for Read 1. In this scenario, remove the entries after Read Number="1" in the RunInfo.xml file.

1. Optional: If you have a SampleSheet.csv file in your run folder, edit the [Reads] section to match the new read assignment in the RunInfo.xml file and import the sample sheet for Local Run Manager v2 and v3 in step 5. Local Run Manager for MiniSeq v1 does not have a sample sheet import option.

2. Copy an RTAComplete.txt file from a successful run folder and paste it into the run folder you are using for analysis.

3. Create a new run in Local Run Manager using the appropriate analysis module and follow the directions for importing data into a new analysis module in the support bulletin Local Run Manager: How to requeue and import run data for reanalysis. If you are using the Local Run Manager interface to set up the new run, enter the modified read length for the last successful read and enter your sample information. For Local Run Manager v2 and v3, you can import the modified sample sheet instead.

4. Analysis will begin automatically after run data import.

For any feedback or questions regarding this article (Illumina Knowledge Article #1807), contact Illumina Technical Support [e-mail].