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 Sample Sheet page for links to additional details on required and optional field inputs for v2 Sample Sheets.

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

Run Planning with Onboard DRAGEN

Run Planning with 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.

  

To ignore an entire index while demultiplexing:

• Use case includes demultiplexing runs that contain both single-indexed and dual-indexed samples

• Remove the "I" from the 'OverrideCycles' setting for the index read that is to be ignored in the [BCLConvert_Settings] section.

• Remove the column (including the column header) for the index read that is to be ignored in the [BCLConvert_Data] section.

  

  

Notes for Manual Sample Sheet Modification

• [Reads] section: This must contain the cycle numbers designated in the RunInfo.xml file (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 completed with 16 cycles for the i5 index, but the actual index for a subset of samples is only 8 bp.

  
• • For some instruments that specify the i5 index in the Reverse Complement orientation, the 'N' is at the beginning of the i5 index (example: Y150;I8;N8I8;Y150). For more information, see Guidelines for reverse complementing i5 sequences for demultiplexing.

• [BCLConvert_Data] section: This section contains the correct indexes.

  • If some index cycles are ignored/treated differently, then this section should contain the correct, shortened indexes.

  • Ns are not recognized as wildcard characters.

  

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