How to avoid Ns in small RNA or short reads when running BCL Convert and bcl2fastq2

Small RNA reads, and reads from other applications where inserts are shorter, are sometimes masked with Ns after FASTQ generation, resulting in the reads being 35 Ns instead of the actual basecalls. N-masking occurs when the length of a read is below the default minimum trimmed read length of the software (35) and the adapter masking length (22) after adapter trimming.

To avoid N-masking when using BCL Convert, add the following two settings in the [BCLConvert_Settings] section of a v2 sample sheet.

MinimumTrimmedReadLength,5 MaskShortReads,5

The application of these settings can be used with standalone BCL Convert, as well as with BCL Convert in BaseSpace and/or Illumina BioInsight Platform Core (formerly, Illumina Connected Analytics (ICA)).

Similarly, to avoid N-masking when using bcl2fastq2, add the following options to the command line.

--minimum-trimmed-read-length 5 --mask-short-adapter-reads 5

More information about these options is available in the bcl2fastq2 Conversion Software v2.20 User Guide.

For any feedback or questions regarding this article (Illumina Knowledge Article #9157), contact Illumina Technical Support [e-mail].