Using an N wildcard in index sequences in different Illumina FastQ generation software

'N' is used as a wildcard character instead of the standard nucleotides (A, C, G, T) in Index sequences. The following reviews the use of the N wildcard in index sequences in different software (bcl2fastq2, MiSeq Reporter, Local Run Manager, and BCL Convert).
MiSeq Reporter and Local Run Manager v2 and v3 allow the use of 'N' as a wildcard character.
bcl2fastq2 and BaseSpace treat 'N' as a literal character (and not a wildcard). Therefore, if there are Ns in the index sequences listed in the sample sheet, bcl2fastq2 and BaseSpace send the associated reads to the Undetermined FastQ file.
  • The use-bases-mask flag can be used with bcl2fastq to mask portions of an index sequence. For example:
    • use --use-bases-mask y*,i*,n*,y* to ignore i5 index
    • or --use-bases-mask y*,i8n*,i*,y*to ignore the last 2 bases of the i7 index.
Likewise, BCL Convert does not treat N as a wildcard character.
  • The OverrideCycles setting can be added to the Settings section of the sample sheet to mask or treat an entire index or part of an index in a different fashion. For example:
    • 'OverrideCycles,Y151;I8U8;I8;Y151' to treat the last 8 bases of the i7 index as a UMI sequence.
For any feedback or questions regarding this article (Illumina Knowledge Article #3560), contact Illumina Technical Support [email protected].
© 2023 Illumina, Inc. All rights reserved. All trademarks are the property of Illumina, Inc. or their respective owners. For specific trademark information, please visit