How to use the 'OverrideCycles' setting in the Run Planning User Interface

Background

The OverrideCycles setting is used to change the way a read or part of read is treated by BCL Convert. The BCL Convert app for onboard DRAGEN or BaseSpace requires a v2 sample sheet, which can be made using Run Planning on select platforms or in BaseSpace.

See the Sample Sheet - Parameters guide for additional details on required and optional field inputs for v2 Sample Sheets.

Setting Override Cycles in the v2 Sample Sheet

Changes must be made to the following sections of the v2 sample sheet while using the 'OverrideCycles' setting.

Key arguments for Override Settings:

• Y - Use data in Read cycles

• U - Use data as UMI cycles

• I - Use data as Index cycles

• N - Do not use data in either Read or Index

Setting Override Cycles in Run Planning

• The Run Planning UI is available in the Control Software on the MiSeq i100 Series and NovaSeq X Series instruments

• BaseSpace now allows for 'OverrideCycles' designation during the Run Planning sample sheet setup.

Onboard DRAGEN

BaseSpace

The 'OverrideCycles' defaults in Run Planning will match the run setup as applicable: Y(Read 1); I(Index 1); I(Index 2): Y(Read 2).

To skip cycles in either the Read or Index, designate the appropriate cycles with the mask flag "N."

• For example, mask the first cycle of each Read for Illumina Stranded RNA libraries using N1Y150 for Reads 1 and 2:

  

If the Index read includes a UMI portion, designate the appropriate cycles with the UMI flag "U."

• For example, a run may have Index 1 with a UMI of 8 bases in a run setup of Read 1 150, Index 1 16, Index 2 8, Read 2 150. The Override Cycles would register as Y150;U8I8;I8;Y150.

  

Manual Sample Sheet Modification

• [Reads] section: This must contain the actual cycle numbers (including the cycles to be ignored or treated differently).

  
• [BCLConvert_Settings] section: This must contain the appropriate 'OverrideCycles' setting.

  
In this example, a sequencing run was set up with 16 cycles for the i5 index, but the actual index for a subset of samples is only 8 bp long.    

For some instruments that specify the i5 index in the Reverse Complement orientation, the 'N' would be at the beginning of the i5 index (Y150;I8;N8I8;Y150).  

• [BCLConvert_Data] section: This section contains the correct indexes; if some index cycles are being ignored/treated differently, then this section should contain the correct, shortened indexes. Ns are not considered as wildcard characters.

  

If an entire index were to be ignored while demultiplexing (eg, when demultiplexing runs with single-indexed and dual-indexed samples), there must be no column (including the column header) for this index read in the [BCLConvert_Data] section.

More information about this setting can be found in the BCL Convert online help page.

For any feedback or questions regarding this article (Illumina Knowledge Article #5671), contact Illumina Technical Support [e-mail].