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

Small RNAs (sRNA) are typically between 21-30 nucleotides in length. Due to their small read lengths, they can become N-masked after adapter trimming. This results in the reads coming 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 of sRNA 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].