Submission Form Guidelines for Direct Sequencing on the NextSeq 2000

Direct sequencing is available only to one group CRUK-CI who have bought their own sequencer. If you know nothing about this, then this page is not for you and you should refer to the Sequencing Only submissions help.

Complete the Pool Information Fields

The direct sequencing form is as small as it can be to allow the processing of the run and delivery of correctly processed files. Many of the fields from the SLX submission form are not necessary if you are setting up the sequencer yourself, though the information may need to be supplied on the sequencer itself. Any problems that come up from operating the sequencer incorrectly are, to be blunt, yours!

Fill out the SLX identifier which is printed on the tube containing your library (SLX-xxxxx).
Select library type from the drop down menu. Some library types have special handling for processing the data, so to get your data easily please pick the correct one.
Important: if submitting for the library types "Amplicon low diversity", "Amplicon high diversity" or "Bisulphite" please read table 3 of the standard sequencing help regarding PhiX spike-in percentage.
Complete billing information. This is critical for correct invoicing: please follow the popup instructions in the field.

Complete the Sample Information Table

Complete a separate row with a sample name and index name for each sample within the pool. For example, a pool of six libraries should use six rows.
1. Sample Name can be anything you choose to represent that sample.
2. Index must be the corresponding Index Name (e.g. i701_i501) rather than the index sequence. Some index types are grouped (for instance all Nextera kits as they all use the same original set of Illumina Nextera index sequence), so as long as the sequence matches your index sequence you are able to submit using that index name. You do not have to fill in a field to state the index type with direct sequencing: it is determined from the indexes you use in your samples. You cannot mix index types though; all indexes used must be of the same type.
3. Species is only needed if you have a mix of species in the pool. Use this field to assign a species for any sample whose species differs from that set in cell C11 (the species common across the pool).
For a pool with In-Line Barcodes or a pool submitted as index type Unspecified (Other), complete a single row for the whole pool, i.e., a pool of six libraries is still only one row in the sample information table. The index should read "INLINE" for index type "inline barcodes" and "Unspecified Index" if "Unspecified (Other)" has been chosen.

Make the Submission

Run your completed submission form through this submission application. Any errors will be reported back to you for correction. If there are no errors, a confirmation page will be displayed with a summary of the submission. Confirming the submission here will create the pools in the Clarity LIMS system ready for sequencing.

Starting Sequencing

This process is the same for newly submitted pools and for additional sequencing of pools that already exist. The practice of creating a new SLX submission in order to run an existing pool a second time is no longer necessary and is very much discouraged.

Find the pool you wish to sequencing from the list of your group's pools. Click on its SLX identifier to view the pool's full details. On this page you will have some additional text and a button that says "Start a sequencing run using this pool". Pressing the button gives you a page where you need to enter the flowcell identifier (not the cartridge id as it was on the NextSeq 500) and the flowcell type. Providing all is well (flowcell id matches the format for the selected flowcell; flowcell hasn't already been used) the system will run the processes in the Clarity LIMS system that have previously been run by hand until the system is in the state where the flowcell is created and populated and the run is ready to start.

That is now all that needs to be done. You will start the run on the sequencer and let it do its job. The LIMS system will automatically read some information from the run folder when it becomes available to properly populate the run process. When the run finishes it will be processed automatically and the run process in the LIMS completed. The run will skip the usual publishing step and go straight to the billing process, which will be the only time members of the Genomics core will need to do anything with the run. The files from the run can be fetched as any other with the download tool.