Troubleshooting FASTQ Generation failing to start automatically in MiSeq using MiSeq Reporter

MiSeq users may encounter FASTQ generation failing to automatically begin in MiSeq Reporter after a run completes. There are several scenarios that may cause this. Review the following scenarios and follow the instructions provided to resolve the given scenario. If the solutions provided do not resolve FASTQ generation, contact Illumina Technical Support for further assistance.

Scenario 1: The sample sheet is not present in the root of the MiSeq Analysis folder

One of the potential cause of missing sample sheet occurs when browsing to a USB drive location for sample sheet during run setup and subsequently remove the USB drive when run starts. This may result in the sample sheet failing to copy to the MiSeqAnalysis folder and prevent FASTQ generation despite RTAComplete.txt present in D:\Illumina\MiSeqAnalysis. Concurrently, there will be no queuedforanalysis.txt or QueuedForAnalysis.txt file to delete in order to requeue.

This may occur if SampleSheet.csv is either not present in the root of the run folder OR has the incorrect file name format.

To resolve this issue:

• Copy the sample sheet into the root of the run folder, make sure the file name is SampleSheet.csv

• Once this is done, the QueuedForAnalysis.txt file will automatically appear and FASTQ generation will begin

• It is best practice to always save the sample sheet file locally and use the locally saved Sample Sheet file path during run setup to ensure sample sheet will be transferred/renamed to the run folders correctly (MiSeq Temp, MiSeq Analysis and MiSeq Output/Network Location)

Scenario 2: queuedforanalysis.txt file nomenclature is incorrect or is not present in the root of the run folder under MiSeq Analysis folder.

This indicates the run was setup without selecting the option "When using BaseSpace or BaseSpace Onsite, replicate analysis locally on MiSeq" in Run Options. This setting enables FASTQ generation locally while saving data to BaseSpace.

Solution:

• Delete the queuedforanalysis.txt file The uppercase version should appear, starting local analysis (QueuedForAnalysis.txt)

• If local analysis is required, ensure the "When using BaseSpace or BaseSpace Onsite, replicate analysis locally on MiSeq" is selected in the MiSeq Control Software during run set-up.

  

Scenario 3: All necessary files appear to be present in the run folder (SampleSheet.csv, RTAComplete.txt), but no version of QueuedForAnalysis.txt or queuedforanalysis.txt is present. MiSeq Reporter (MSR) service is running, but analysis does not start.

The most common cause for this scenario is a sample sheet that has been edited such that MSR does not recognize the file as valid input and cannot parse it. Determining the specific element in the sample sheet that is causing a problem will resolve this issue.

Solution:* Test if the sample sheet by renaming or removing the current SampleSheet.csv file from the run folder. Copy the SampleSheet.csv file from a previous successful run into the run folder and determine if analysis starts (QueuedForAnalysis.txt should appear if analysis begins successfully).

• This analysis may then be terminated from the MSR interface. Then create a new sample sheet or sample sheet template within Illumina Experiment Manager to ensure proper sample sheet formatting.

• Copy the new sample sheet (with the name SampleSheet.csv) to the run folder and delete QueuedForAnalysis.txt to restart analysis with the corrected sample sheet.

For any feedback or questions regarding this article (Illumina Knowledge Article #3614), contact Illumina Technical Support [e-mail].