The Illumina Stranded mRNA protocol is not strongly stranded for mRNA with low U content

The Illumina TruSeq Stranded and Illumina Stranded mRNA protocols are commonly used for strand-specific bulk RNA-seq and they typically yield >99% antisense reads. We show that these protocols can generate sense-oriented reads for transcripts with extremely low U content (<3%). Indeed, such regions can bypass the dUTP-based blockade of cDNA second strand amplification. A small number of genes are affected by this issue (three in Drosophila melanogaster , including the glue gene Sgs3 , and 46 in Mus musculus ). To prevent overestimation of expression levels, we recommend excluding sense reads for all genes.