How to generate FASTQ files from an incomplete run in MiSeq Reporter

It is possible to generate FASTQ files, even if a MiSeq run does not complete. To do so, determine the last cycle with a complete set of *.bcl files, modify run files, and requeue analysis. This bulletin describes how to generate FASTQ files from an incomplete run using MiSeq Reporter. If you are using Local Run Manager instead of MiSeq Reporter see the bulletin Local Run Manager: Generate FASTQ files for an incomplete run.

Before you start:* If you plan to do secondary analysis on the MiSeq, make sure that a sequencing run is not in progress and that MiSeq Reporter is ready for analysis.

• 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 (ie, in Read 1), it is not possible to demultiplex your samples.

• Make sure that the RTAComplete.txt file is not present in the run folder.

Steps to take:

1. Navigate to D:\Illumina\MiSeqAnalysis[runfolder]\Data\Intensities\BaseCalls\L001 and check which cycle was the last to generate all *.bcl files. If v3 reagents were used, there are 38 *.bcl files per cycle. If v2 reagents were used, there are 28 *.bcl files per cycle.

*If .bcl files are not present, it might be possible to recover them by manually launching RTA (Real-Time Analysis). To do so, contact Illumina Technical Support.

Calculate how many cycles have been successfully completed and extracted.

• For example:

  • Run stopped at cycle 207 in a 2 x 150 + 6 bp index run. If the last cycle with complete *.bcl files was cycle 206, it is a good rule of thumb not to push against the last cycle with complete *.bcl files. Select the previous complete cycle from which to do the analysis. In this case, it is cycle 205 . The run breakdown is (R1) 150 + (R2 Index) 6 + (R3) 49 = 205 cycles.

1. Backup the original RunInfo.xml and SampleSheet.csv files by renaming them to something like RunInfo.xml.BAK and SampleSheet.csv.BAK. Using Notepad, edit these documents and save the edited documents as RunInfo.xml and SampleSheet.csv in the root run folder in MiSeqAnalysis. If the reverse read of a paired-end run - Read 3 in the following example - has not been performed, delete the corresponding line in RunInfo.xml and SampleSheet.csv.

• For Example:

RunInfo.xml:

SampleSheet.csv:

1. Copy an RTAComplete.txt file from a successful run folder and paste it into your run folder.

2. You can expect to see the run queued in MiSeq Reporter and a QueuedForAnalysis.txt file created in the run folder in MiSeqAnalysis. To make sure that the run has been requeued in MiSeq Reporter, point a web browser window to http://localhost:8042 and look for your run in the list opened with the Analyses button.

For any feedback or questions regarding this article (Illumina Knowledge Article #1325), contact Illumina Technical Support [e-mail].