Troubleshooting FASTQ generation not occurring automatically after a MiSeq run has completed successfully and successfully uploaded to BaseSpace

In some instances, FASTQ generation will not automatically occur in BaseSpace upon successful completion of the run on the instrument and the run uploading to BaseSpace.

The root cause of this is most often either a sample sheet error or the data folder not being included in the upload as a result of the run setup mode.

During MiSeq run setup, there are 2 options available for uploading data to BaseSpace:

1. Run Monitoring from anywhere

• This setting is commonly referred to as 'Run Monitoring ONLY' and will send only run information files to BaseSpace Sequence Hub so runs can be monitored remotely.

1. Run analysis, collaboration, and storage

• This setting is commonly referred to 'Run Monitoring AND STORAGE' and will send all run data to BaseSpace Sequence Hub for remote monitoring, data analysis, collaboration, and storage.

Selecting 'Run Monitoring from anywhere' means that the BCL files will not be included in the run upload and thus the software will be unable to generate FASTQ files.

Identifying the problem

This can be identified in one of two ways.

1. Review the RunParameters.xml file located in the MiSeqTemp folder (D:\Illumina\MiSeqTemp)

   1. Locate

      1. The run was set up as Run Monitoring ONLY if the mode is listed as "IpdRequested RunMonitoringRequested"

      2. The run was set up as Run Monitoring AND STORAGE if the mode is listed as "IpdRequested RunMonitoringRequested RunStorageRequested"

2. Review the files listed in BaseSpace

   1. Select the run in question on BaseSpace

   2. Select the 'FILES' tab

   3. Select the 'Data' folder

   4. Select the 'Intensities' folder

   5. Select the 'BaseCalls' folder

      1. If there are only folders titled 'Matrix' and 'Phasing' (and there is no 'L001' folder present), the run was uploaded as run monitoring only

      2. As a workaround, the run will need to be re-uploaded to BaseSpace in order to be demultiplexed on BaseSpace (instructions listed in the 'Workaround' section below).

      3. If there is an 'L001' folder present within the BaseCalls folder, select that folder and confirm all expected data cycles are present in said folder, and contact Illumina Technical Support for further assistance in troubleshooting.

      4. An example of a run folder that shows BCL files is shown below:

Workaround

As a workaround, the run folder can be uploaded to BaseSpace using the BaseSpace Command Line Interface (CLI), ensuring that the data folder is included in the subset of files being uploaded to BaseSpace. For the MiSeq specifically, be sure to upload run files from the MiSeqAnalysis or MiSeqOutput folder, as the MiSeqTemp folder does not contain BCL files.

Instructions for uploading the run via BaseSpace CLI can be found here: Instructions for using BaseSpace CLI on Windows to upload runs

Preventing this from occurring To avoid this issue in the future, runs should be set up as 'Run analysis, collaboration, and storage'. This will ensure the data folder is included at the time of run upload and as long as the SampleSheet.csv, InterOp, RunInfo.xml, and RunParameters.xml files are present, the run should begin demultiplexing automatically upon completion of the upload to BaseSpace.

See the following instructions for Enabling run storage and analysis in BaseSpace for MiSeq runs

For any feedback or questions regarding this article (Illumina Knowledge Article #8692), contact Illumina Technical Support [e-mail].