TruSeq Stranded RNA Kit FAQs: Sequencing settings

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 guidance on some factors to consider - note that these recommendations are based on internal and external data, but do not represent strict cut-offs. Needs for individual projects may vary based on multiple variables as well as user preference.

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.

Why is there bias sequencing the ends of stranded RNA molecules? Since the reads are directional and the average fragment size is larger than the recommended read-length, read 1 has better coverage at the 3' end, while read 2 has better coverage at the 5' end. Consequently, we recommend paired-end reads.

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