Illumina RNA Prep Tagmentation (L) with Enrichment Sequencing FAQs

The following are commonly asked questions for sequencing Illumina RNA Prep Tagmentation (L) with Enrichment Sequencing libraries.

What is the recommended read length and number of reads per sample?

• Illumina recommends a paired-end run of 76 cycles per read (2x76) with 10 cycles for each index read (Read 1: 76, Index 1: 10, Index 2: 10, Read 2: 76) regardless of the enrichment probe panel or sample input type.

• Libraries can be sequenced with longer read lengths (2x101, 2x126 or 2x151) if a lab would like overlapping reads or additional raw coverage, but it is not required.

What are the advantages of performing paired-end sequencing of RNA libraries? Paired-end sequencing helps with data alignment, discovery of novel transcripts, and identification of PCR duplicates.

What are the suggested loading concentrations for the various sequencers? The Illumina RNA Prep with Enrichment Reference Guide includes select loading concentration recommendations. For other sequencers and panels, consult the denature and dilution guide of the sequencer that will be used.

Can libraries prepared with this kit be pooled with other library types for sequencing? This is not supported and results are not guaranteed.

What is the recommended sequencing depth/number of reads per sample? Generally, 3-25 million reads per sample, depending on the panel. The read requirement depends on the enrichment panel used and data needs for each experiment, and may need to be optimized.

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