How to avoid Ns in small RNA reads or short reads after running bcl2fastq2?

Small RNA reads 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 read length is below the default minimum trimmed read length (ie, 35) and the adapter masking length (ie, 22) after adapter trimming.

To avoid N-masking in the reads, add the following to the bcl2fastq2 command line:

--minimum-trimmed-read-length 5

--mask-short-adapter-reads 5

More information about these options can be found in the bcl2fastq2 Conversion Software v2.20 User Guide.

For any feedback or questions regarding this article (Illumina Knowledge Article #3147), contact Illumina Technical Support [e-mail].