Fig. 1

Differential transcriptome sampling by polyA+ and ribo-minus RNA selection methods leads to variance in TPM estimates. Each point is a gene, coloured by type: black points represent protein-coding genes, pseudogenes and processed pseudogenes (n = 21,211); blue points represent RNA genes (n = 5843). The line y = x is shown in red. As ribo-minus libraries capture RNA genes that polyA+ libraries do not (the line of points at x = 0), expression can be systematically underestimated for the remaining, mostly protein-coding, genes (Fig. 2). The data shown is for BMDMs prior to LPS stimulation. The same pattern was observed for BMDMs 7 h post-LPS stimulation (Additional file 1: Figure S1). The confounding effect of selection method can be corrected mathematically (Fig. 4)