Sequencing depth and read length considerations for TruSeq Stranded RNA libraries

What is the recommended run length and type for these libraries? The read length and format (single read versus paired end) are important considerations in the design of RNA sequencing experiments. The table below provides general guidance on some factors to consider - note that these are common configurations and read lengths, but not official recommendations. Needs for individual projects may vary based on multiple variables as well as user preference. M = million.

Applications	Read Type	Read depth / sample (mRNA / total RNA)
Gene profiling (gene-level counts)	1 x 50	>5M / >10M
Discovery (alternative transcripts, gene fusions, etc)	2 x 50 - 2 x 75	≥50M / >100M
Complete transcripome annotation	2 x 75 - 2 x 100	≥100M / ≥200M

Why does Illumina recommend paired-end sequencing of stranded RNA libraries? Due to the directional nature of the assay, paired-end sequencing captures both ends of the RNA molecule, and allows for assessing true duplicate rates.

For any feedback or questions regarding this article (Illumina Knowledge Article #1151), contact Illumina Technical Support [e-mail].