QC steps FAQ for Illumina Rapid RNA Kits

Is there any change to the expected library QC for Rapid kits compared to their non-Rapid versions?

Final Library Size (observed by TapeStation or Bioanalyzer):

• mRNA: Illumina Rapid mRNA and Illumina Stranded mRNA have similar library sizes in the ~300-400 bp range.

• Total RNA & RNA with Enrichment: Rapid libraries are smaller in size compared to Illumina Stranded Total RNA or Illumina RNA Prep with Enrichment libraries, respectively. (The distributions will be shifted to the left when viewed in library QC traces).

What is the average insert size for Rapid RNA preps?

120-150 bp. The average Rapid Total RNA library prep size is about 250 bp, which means the insert size is about 130 bp.

Is it possible to adjust fragmentation time for Rapid RNA, similar to the TruSeq Stranded RNA kits, to create libraries with longer fragment length for use with 1000 cycle sequencing kits?

No, the Rapid RNA preps have shorter inserts of ~200 bp after library prep due to EBLTS, which when sequenced on XLEAP-SBS are ~120-160 bp average aligned insert size, and even shorter (<100 bp) on MiSeq i100. It is not possible to adjust the insert size and attempts to size select the libraries with Illumina Purification Bead (IPB) clean up would result in loss of library yield and potentially biased coverage of the RNA expression.

For any feedback or questions regarding this article (Illumina Knowledge Article #10214), contact Illumina Technical Support [e-mail].