How to requeue analysis of a NextSeq 1000/2000 run in BaseSpace Sequence Hub

The Illumina NextSeq 1000/2000 sequencing system offers options for sequencing data analysis, through DRAGEN onboard or in the cloud with data uploaded to BaseSpace Sequence Hub. Run configuration and analysis options for NextSeq 1000/2000 runs uploaded to BaseSpace Sequence Hub are set up through Run Planning. Sample or index information for NextSeq 1000/2000 runs in BaseSpace can be corrected for reanalysis through a different process than other sample sheet-based runs. This article explains how to reanalyze a NextSeq 1000/2000 run in BaseSpace Sequence Hub. How a customer requeues is based off what version of DRAGEN they are using.

Creating a corrected sample sheet

Analysis of a NextSeq 1000/2000 run in BaseSpace Sequence Hub requires a correctly formatted v2 sample sheet. Using BaseSpace Run Planning is recommended to generate a sample sheet for reanalysis to make sure that the required fields are correct. Other sample sheet generation tools such as Illumina Experiment Manager or Local Run Manager do not create v2 sample sheet files. The v2 sample sheet template on the NextSeq 1000/2000 support site can also be used. However, the template does not contain the Cloud_Data section used in project selection that is discussed in the following section.

Steps for creating a v2 sample sheet in Run Planning for reanalyzing a run:

1. After logging in to BaseSpace Sequence Hub, select the Runs tab.

2. Select New Run, and then select Run Planning from the dropdown menu.

1. In the Run Settings > Secondary Analysis field, either BaseSpace or Local can be selected as the analysis location. Both allow for sample sheet export.

2. In the Configuration > Application field, select Illumina DRAGEN FASTQ Generation. Only FASTQ generation can be requeued in BaseSpace. If reanalysis with any of the other DRAGEN workflows is required, generate FASTQs first and then launch the appropriate DRAGEN app in BaseSpace.

3. Enter in the necessary library, index, and sample information in the two Configuration screens.6. In the Analysis Setting section, enter in any of the optional information, if needed.

4. Export the sample sheet after the run setup is complete:

• If the Analysis location was set as BaseSpace, return to the Runs tab, and select Planned.

  
• Check the box next to the planned run, then select the File button, Download, and Sample Sheet.

  
• If the Analysis Location was set as Local, select the Export near the bottom right of the screen to download the SampleSheet.csv file. If Submit Run is selected instead, follow steps for BaseSpace, above, to download the sample sheet from the planned run.

  

Important Notes:

• Run names and Sample IDs in v2 sample sheets cannot contain spaces or special characters; use only letters, numbers, dashes, or underscores.

• Projects can be designated for output data by using the ProjectName in the Cloud_Data section of the sample sheet. If no ProjectName value is set, then the output project will use the value from the RunName entry in the sample sheet header.

• Manual edits to the exported sample sheet before use in analysis are discouraged. Incorrect, missing, or unexpected entries can cause analysis failure.

Reanalyzing the run data

Reanalysis and regeneration of FASTQs from NextSeq 1000/2000 run data in BaseSpace requires the use of the BCL Convert app. Attempting to requeue analysis from the run Status menu will return an error. Follow these steps to reanalyze a NextSeq 1000/2000 run with the BCL Convert app:

1. Upload the corrected sample sheet to the project by selecting Upload then Files. Select the Other file type and import the sample sheet CSV file.

2. Navigate to the BCL Convert app page.3. Select Launch Application from the app page.

3. Select Runs to choose a run from the BaseSpace Sequence Hub account.

4. Under Override Sample Sheet, use Select Dataset File(s) to select the sample sheet uploaded in step 1. Launching the BCL Convert app without a new sample sheet will use the previous sample sheet already in the run folder.

5. Select Launch Application.

For any feedback or questions regarding this article (Illumina Knowledge Article #7474), contact Illumina Technical Support [e-mail].