Fig. 1

Main challenges in total-RNA-seq quantification. a Integration of databases from multiple sources (indicated in orange, green and cyan) can lead to redundant annotations of the same feature with different names, formats or slightly different coordinates. b Example of a multi-mapping read (black line) that aligns equally well to more than 1 position (yellow lines) and cannot be assigned to a genomic origin unequivocally. c Example of a multi-overlapping read that maps to a genomic position where two annotated features coexist (a mRNA exon and a snoRNA) and cannot be assigned to a feature unequivocally. d Examples of the heterogeneous relations between genomic loci and transcribed molecules integrated in total-RNA-seq: a cluster of small-RNA loci (e.g. pi6, from the piRNA biotype) can actively transcribe the same product simultaneously; microRNA transcripts result in two distinct transcripts (e.g. unprocessed mi8 is post-clipped to 3p/5p mature forms); long molecules comprising exons and introns undergo splicing, resulting in reads from precursor and mature transcripts