Sequencing FAQs for Illumina Stranded RNA libraries

The information in this article pertains specifically to the Illumina Stranded mRNA and the Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus (or Ribo-Zero Plus Microbiome) library prep kits.

What are the advantages of performing paired-end sequencing of RNA libraries?

Paired-end sequencing can help with data alignment, discovery of novel transcripts, and identification of PCR duplicates.

What are the suggested loading concentrations for the various sequencers?

Recommended loading concentrations can be found in either the Illumina Stranded mRNA Reference Guide, Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus Reference Guide, or Denature and Dilute Protocol Generator workflow for each instrument. Loading concentrations may require optimization within each lab.

Note that Illumina Stranded RNA libraries require dark cycles recipes or sufficient PhiX spike-in during sequencing. See the following resources for more information:

• Custom recipes for Illumina Stranded libraries on NextSeq 1000/2000

• Custom recipes for Illumina Stranded Total RNA and mRNA libraries on NovaSeq X Series

• PhiX requirements for sequencing Illumina Stranded RNA libraries

Which strand is sequenced in Read 1?

Read 1 sequences will be identical to the antisense strand of DNA, which is also the first strand cDNA.

Can libraries prepared with this kit be combined with other library types in the same flow cell lane?

This is not supported.

For any feedback or questions regarding this article (Illumina Knowledge Article #3291), contact Illumina Technical Support [e-mail].