# Using an N wildcard in index sequences in different Illumina FASTQ generation software

The N character is sometimes used as a wildcard to indicate "any base" instead of the standard bases (A, C, G, T) in index sequences, however not all demultiplexing and FASTQ generation software treats an N the same way. The following information reviews the use of the N character in index sequences in different Illumina software (MiSeq Reporter, Local Run Manager, bcl2fastq2, and BCL Convert).

**MiSeq Reporter** and **Local Run Manager** v2, v3, and v4 allow the use of N as a wildcard character, where the N can stand in for and match to any standard base. This is frequently used when the bases are unknown or when attempting to include indexes of different lengths in the same analysis, known as N-padding.

**bcl2fastq2** and **BCL Convert** treat N as a literal character/basecall (and not a wildcard). Therefore, if there are Ns in the index sequences listed in the sample sheet, bcl2fastq2 and BCL Convert will attempt to match Ns in the sample sheet to the bases read during the indexing cycles. Depending on the number of mismatches allowed when configuring the software, using Ns may send the associated reads to the Undetermined FATSQ file instead of the intended sample FASTQs.

In order to correctly mask bases in these software:

* In bcl2fastq the --**use-bases-mask** flag can be used along with a sample sheet containing the correct number of bases in the index columns to mask portions of an index sequence. For example:
  * Using **--use-bases-mask y\*,i\*,n\*,y\*** will demultiplex using the entire i7 index and ignore the entire i5 index.
  * Using **--use-bases-mask y\*,i8n\*,i\*,y\***will demultiplex using the first 8 cycles of the i7 index and ignore any remaining bases in the i7 index, and use the entire i5 index.
* In BCL Convert a similar functionality is achieved by using the OverrideCycles setting in a sample sheet along with the correct number of bases in the index columns to mask or treat an entire index or part of an index. For example:
  * Using OverrideCycles,Y151;I8;N8;Y151 in a dual-indexed run with 8 cycles in each index will demultiplex using the entire i7 index and the ignore the entire i5 index.
  * Using OverrideCycles,Y151;I8N2;I10;Y151 in a dual-indexed run with 10 cycles in each index will use demultiplex using the first 8 cycles of the i7 index and ignore the last two cycles, and use all 10 cycles of the i5 index.
* For information on handling UMI cycles in index reads with BCL Convert, see [BCL Convert and index UMIs - How to demultiplex samples and output a UMI FASTQ](https://knowledge.illumina.com/software/on-premises-software/software-on-premises-software-reference_material-list/000007337). bcl2fastq does not support the use of UMIs in index reads and neither MiSeq Reporter nor Local Run Manager support any UMI usage in FASTQ Generation.

Important note: bcl2fastq supports the use of the **\*** (asterisk)character in the `--use-bases-mask` option to mean "all remaining cycles", while BCL Convert requires OverrideCycles to explicitly define how each cycle is being used. BCL Convert does not allow the use of the asterisk character in OverrideCycles.

Related articles:

* [How to demultiplex a sequencing run without using either the i7 or the i5 index using BCL Convert](https://knowledge.illumina.com/software/on-premises-software/software-on-premises-software-reference_material-list/000005166)
* [Options for demultiplexing libraries with different index lengths in the same sequencing run](https://knowledge.illumina.com/software/general/software-general-troubleshooting-list/000005982)

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