Illumina Stranded mRNA Prep Ligation Sequencing FAQs

What is the recommended read length and number of reads?

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, but do not represent strict cut-offs or official recommendations. Needs for individual projects may vary based on multiple variables as well as user preference. Consult the literature for the most up to date resources. (M = million)

Application	Read Type	Read Depth per sample (mRNA/Total RNA)
Gene profiling (gene-level counts)	1 x 51	>5 M / >10 M
Differential gene expression	2 x 76	30-60 M
Discovery (alternative transcripts, gene fusions, etc.)	2 x 76	≥50 M / >100 M
Complete transcriptome annotation	2 x 76-101	≥100 M / ≥200 M

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?

Follow the guidelines in the specific reference guides of the instrument; loading concentrations may require optimization within each lab.

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.

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