Sequencing and run setup FAQ for Illumina Rapid RNA Kits

What are the recommended read depths or recommended number of samples per run for Illumina Rapid RNA kits? Is this different than their non-Rapid versions? What is the recommended read length for sequencing?

See the article Read Depth Requirements for Illumina Rapid RNA kits.

Why do Rapid libraries need shorter read lengths and fewer sequencing reads compared to legacy kits?

The shorter recommended read length compared to prior RNA-Seq workflows is to enable RNA sequencing at a lower cost (via a smaller cycle kit). The Rapid kits are generally recommended to be sequenced at 100 total cycles (2x50 or 1x100). This does not affect mapping, and therefore gene expression counting, compared to previous longer read lengths, but it could affect the discovery of variants/variant calling. Also, duplicate rates are generally higher when not sequencing paired-end (PE) reads.

Generally, with increased sequencing depth, there are diminishing returns on the number of genes detected. Rapid RNA datasheets contain figures showing the number of genes detected and duplicate rate as functions of increased sequencing depth. For most applications, sufficient mRNA reads are obtained at 10 million and 20 million read (cluster) depth, and at 25 million and 50 million read (cluster) depth for Total RNA.

Do Rapid RNA libraires require a custom sequencing recipe or dark cycle recipe?

Custom sequencing recipes or dark cycle recipes are not required to sequence Rapid libraries.

Are there any recommended override cycles for Rapid kits?

No override cycles are required for Rapid kits.

What is the adapter trimming sequence for Illumina Rapid RNA kits?

CTGTCTCTTATACACATCT

How much PhiX is recommended for Illumina Rapid RNA libraries?

As the Rapid workflows do not change the relative base diversity of the library prep kit, the same amount of PhiX as used for non-Rapid workflows can be used with their Rapid versions.

Notably, Illumina Stranded RNA kits need either a dark cycle sequencing recipe or sufficient PhiX spike-in to mitigate Read1 and Read2 beginning with 100% T-base (details here). In Illumina Rapid mRNA and Rapid Total workflows, this 100% T-base is not present at the start of Read1 and Read2, so dark cycle sequencing recipes or higher PhiX spike-in are not required.

Is BaseSpace Run Planning updated with these new Rapid library kits?

BaseSpace Sequence Hub (BSSH) will be updated to include these kits:

• New library prep kits:

• Illumina Rapid mRNA Prep (Default read lengths: 2 x 51)

• Illumina Rapid Total RNA Prep (Default read lengths: 2 x 51)

• Illumina Rapid RNA Prep with Enrichment (Default read lengths: 2 x 101)*

• Illumina Rapid RNA Prep with Exome 2.5 Enrichment (Default read lengths: 2 x 101)

• Compatible index adapter kits:

• Illumina DNA/RNA UD Indexes Set A Tagmentation (v3)

• Illumina DNA/RNA UD Indexes Set B Tagmentation (v3)

• Illumina DNA/RNA UD Indexes Set C Tagmentation (v3)

• Illumina DNA/RNA UD Indexes Set D Tagmentation (v3)

• Illumina DNA/RNA UD Indexes Set A B C D Tagmentation (v3)

* Note: choose Illumina Rapid RNA Prep with Enrichment for the following kits:

• Illumina Rapid RNA Prep with Enrichment (RRPE)

• Illumina Rapid RNA Prep with Exome 2.5 Enrichment (Rapid Exome 2.5)

• Illumina Rapid Viral Surveillance Panel v2 (Rapid VSPv2)

• Illumina Rapid Respiratory Pathogen ID/AMR Enrichment Panel Kit (Rapid RPIP)

• Illumina Rapid Respiratory Virus Enrichment Kit (Rapid RVEK)

Library prep kits are available for all versions of DRAGEN RNA and BCL Convert.

For any feedback or questions regarding this article (Illumina Knowledge Article #10215), contact Illumina Technical Support [e-mail].